Transcriptional activator nucleic acids, polypeptides and methods of use thereof

ABSTRACT

The invention provides isolated nucleic acids and their encoded proteins which act as transcriptional activators and methods of use thereof. The invention further provides expression cassettes, transformed host cells, transgenic plants and plant parts, and antibody compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a divisional application that claims priority to U.S.application Ser. No. 09/435,054 filed Nov. 8, 1999; U.S. ProvisionalApplications Serial No. 60/107,643 filed Nov. 9, 1998 and U.S.Provisional Application No. 60/107,810 filed Nov. 10, 1998.

TECHNICAL FIELD

[0002] The present invention relates generally to plant molecularbiology. More specifically, it relates to nucleic acids and methods formodulating their expression in plants.

BACKGROUND OF THE INVENTION

[0003] Major advances in plant transformation have occurred over thelast few years. However, in major crop plants, such as maize andsoybeans, serious genotype limitations still exist. Transformation ofagronomically important maize inbred lines continues to be bothdifficult and time consuming. Traditionally, the only way to elicit aculture response has been by optimizing medium components and/or explantmaterial and source. This has led to success in some genotypes, but mostelite hybrids fail to produce a favorable culture response. While,transformation of model genotypes is efficient, the process ofintrogressing transgenes into production inbreds is laborious, expensiveand time consuming. It would save considerable time and money if genescould be introduced into and evaluated directly in commercial hybrids.

[0004] Current methods for genetic engineering in maize require aspecific cell type as the recipient of new DNA. These cells are found inrelatively undifferentiated, rapidly growing callus cells or on thescutellar surface of the immature embryo (which gives rise to callus).Irrespective of the delivery method currently used, DNA is introducedinto literally thousands of cells, yet transformants are recovered atfrequencies of 10⁻⁵ relative to transiently-expressing cells.Exacerbating this problem, the trauma that accompanies DNA introductiondirects recipient cells into cell cycle arrest and accumulating evidencesuggests that many of these cells are directed into apoptosis orprogrammed cell death. (Reference Bowen et al, Third InternationalCongress of the International Society for Plant Molecular Biology, 1991,Abstract 1093). Therefore it would be desirable to provide improvedmethods capable of increasing transformation efficiency in a number ofcell types.

[0005] Typically a selectable marker is used to recover transformedcells. Traditional selection schemes expose all cells to a phytotoxicagent and rely on the introduction of a resistance gene to recovertransformants. Unfortunately, the presence of dying cells may reduce theefficiency of stable transformation. It would therefore be useful toprovide a positive selection system for recovering transformants.

[0006] In spite of increases in yield and harvested area worldwide, itis predicted that over the next ten years, meeting the demand for cornwill require an additional 20% increase over current production(Dowswell, C. R., Paliwal, R. L., Cantrell, R. P., 1996, Maize in theThird World, Westview Press, Boulder, Colo.).

[0007] In hybrid crops, including grains, oil seeds, forages, fruits andvegetables, there are problems associated with the development andproduction of hybrid seeds. The process of cross-pollination of plantsis laborious and expensive. In the cross-pollination process, the femaleplant must be prevented from being fertilized by its own pollen. Manymethods have been developed over the years, such as detasseling in thecase of corn, developing and maintaining male sterile lines, anddeveloping plants that are incompatible with their own pollen, to name afew. Since hybrids do not breed true, the process must be repeated forthe production of every hybrid seed lot.

[0008] To further complicate the process, inbred lines are crossed. Forexample in the case of corn, the inbreds can be low yielding. Thisprovides a major challenge in the production of hybrid seed corn. Infact, certain hybrids cannot be commercialized at all due to theperformance of the inbred lines. The production of hybrid seeds isconsequently expensive, time consuming and provides known and unknownrisks. It would therefore be valuable to develop new methods whichcontribute to the increase of production efficiency of hybrid seed.

[0009] As new traits are added to commercial crops by means of geneticengineering, problems arise in “stacking” traits. In order to developheritable stacked traits, the traits must be linked because ofsegregating populations. Improved methods for developing hybrid seedwhich would not require linking of the traits would significantlyshorten the time for developing commercial hybrid seeds.

[0010] Gene silencing is another problem in developing heritable traitswith genetic engineering. Frequently gene silencing is seen followingmeiotic divisions. Elimination or reduction of this problem wouldadvance the state of science and industry in this area.

SUMMARY OF THE INVENTION

[0011] It is the object of the present invention to provide nucleicacids and polypeptides relating to embryogenesis.

[0012] It is another object of the present invention to provide nucleicacids and polypeptides that can be used to identify interacting proteinsinvolved in transcription regulation in embryogenesis.

[0013] It is another object of the present invention to provideantigenic fragments of the polypeptides of the present invention.

[0014] It is another object of the present invention to providetransgenic plants and plant parts containing the nucleic acids of thepresent invention.

[0015] It is another object of the present invention to provide methodsfor modulating, in a transgenic plant, the expression of the nucleicacids of the present invention.

[0016] It is another object of the present invention to provide a methodfor improving transformation frequencies.

[0017] It is another object of the present invention to provide a methodfor improving transformation efficiency in cells from various sources.

[0018] It is another object of the present invention to provide a methodfor a positive selection system.

[0019] It is another object of the present invention to provide a methodfor efficiently producing hybrid seed via apomixis.

[0020] It is another object of the present invention to provide a methodfor stacking traits which does not require linking of traits.

[0021] It is another object of the present invention to provide a methodfor reducing the problem of gene silencing.

[0022] The present invention relates to a HAP3-type CCAAT-box bindingtranscriptional activator polynucleotides and polypeptides, and inparticular, the leafy cotyledon 1 transcriptional activator (LEC1)polynucleotides and polypeptides. In other aspects the present inventionrelates to expression cassettes optionally linked in antisenseorientation, host cells transfected with at least one expressioncassette, and transgenic plants and seeds comprising the expressioncassettes. Further aspects of the invention include methods of using thepolynucleotides and polypeptides. In a further aspect, the presentinvention relates to a method of modulating expression of thepolynucleotides encoding the polypeptides of the present invention in aplant. Expression of the polynucleotides encoding the proteins of thepresent invention can be increased or decreased relative to anon-transformed control plant.

BRIEF DESCRIPTION OF THE DRAWING

[0023]FIG. 1 depicts the comparison of various sequences and thealignment of the conserved regions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0024] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in its naturally occurring environment or (2) if the material isin its natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in the cellother than the locus native to the material.

[0025] As used herein, “nucleic acid” means a polynucleotide andincludes single or double-stranded polymer of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include modifiednucleotides that permit correct read through by a polymerase and do notalter the expression of a polypeptide encoded by the polynucleotide.

[0026] As used herein, “LEC1 nucleic acid” means a nucleic acid orpolynucleotide that codes for a LEC1 polypeptide.

[0027] As used herein, “polypeptide” means proteins, protein fragments,modified proteins, amino acid sequences and synthetic amino acidsequences. The polypeptide can be glycosylated or not.

[0028] As used herein, “LEC1 polypeptide” means a HAP3 family member,CCAAT-box binding transcriptional activator polypeptide that regulatesgene expression during embryo development, and that contains theconserved sequence set out in SEQ ID NO: 23.

[0029] As used herein, “plant” includes plants and plant parts includingbut not limited to plant cells, plant tissue such as leaves, stems,roots, flowers, and seeds.

[0030] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.

[0031] By “fragment” is intended a portion of the nucleotide sequence ora portion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native nucleic acid.Alternatively, fragments of a nucleotide sequence that are useful ashybridization probes generally do not encode fragment proteins retainingbiological activity. Thus, fragments of a nucleotide sequence aregenerally greater than 20, 30, 50, 100, 150, 200 or 300 nucleotides andup to the entire nucleotide sequence encoding the proteins of theinvention. Generally the probes are less than 1000 nucleotides andpreferably less than 500 nucleotides. Fragments of the invention includeantisense sequences used to decrease expression of the inventivepolynucleotides. Such antisense fragments may vary in length rangingfrom at least about 20 nucleotides, about 50 nucleotides, about 100nucleotides, up to and including the entire coding sequence.

[0032] By “functional equivalent” as applied to a polynucleotide or aprotein is intended a polynucleotide or a protein of sufficient lengthto modulate the level of LEC1 protein activity in a plant cell. Apolynucleotide functional equivalent can be in sense or antisenseorientation.

[0033] By “variants” is intended substantially similar sequences.Generally, nucleic acid sequence variants of the invention will have atleast 60%, 65%, or 70%, preferably 75%, 80% or 90%, more preferably atleast 95% and most preferably at least 98% sequence identity to thenative nucleotide sequence, wherein the % sequence identity is based onthe entire sequence and is determined by GAP analysis using Gap Weightof 50 and Length Weight of 3. Generally, polypeptide sequence variantsof the invention will have at least about 50%, 55%, 60%, 65%, 70%, 75%or 80%, preferably at least about 85% or 90%, and more preferably atleast about 95% sequence identity to the native protein, wherein the %sequence identity is based on the entire sequence and is determined byGAP analysis using Gap Weight of 12 and Length Weight of 4.

[0034] A “responsive plant cell” or “responsive host cell” refers to acell that exhibits a positive response to the introduction of LEC1polypeptide or LEC1 polynucleotide compared to a cell that has not beenintroduced with LEC1 polypeptide or LEC1 polynucleotide. The responsecan be to enhance tissue culture response, induce somatic embryogenesis,induce apomixis, increase transformation efficiency or increase recoveryof regenerated plants.

[0035] A “recalcitrant plant cell” is a responsive plant cell thatgenerally does not exhibit a positive response such as tissue cultureresponse, transformation efficiency or recovery of regenerated plants.

Nucleic Acids

[0036] The present invention relates to a HAP3-type CCAAT-box bindingtranscriptional activators, and in particular, the leafy cotyledon 1transcriptional activator (LEC1). Expression of the LEC1 polynucleotideinitiates formation of embryo-like structures and improves growth andrecovery of transformants. The term apomixis is used to describe asexualreproduction that replaces or substitutes sexual methods ofreproduction. When apomixis occurs, embryos are produced from maternaltissue and use only the maternal genome.

[0037] In particular the present invention relates to an isolatednucleic acid comprising a member selected from the group consisting of:

[0038] (a) a polynucleotide which encodes a polypeptide of SEQ ID NO: 2,8, 10, 12, 14, 16, 18, 20, or 22;

[0039] (b) a polynucleotide amplified from a plant nucleic acid libraryusing the primers of SEQ ID NOS: 3 and 4, 5 and 6, 9 and 10, or 11 and12 or primers determined by using Vector nti Suite, InforMax Version 5.

[0040] (c) a polynucleotide comprising at least 20 contiguous bases ofSEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19, or 21;

[0041] (d) a polynucleotide encoding a plant HAP3-type ccaat-boxtranscriptional activator with the conserved motif of SEQ ID NO: 23,wherein the polynucleotide is from a plant other than Arabidopsis;

[0042] (e) a polynucleotide having at least 60% sequence identity to SEQID NO: 1, 9, 11, 13, 17, or 21 or 65% sequence identity to SEQ ID NO: 15or 19 or 70% sequence identity to SEQ ID NO: 7, wherein the % sequenceidentity is based on the entire sequence and is determined by GAPanalysis using Gap Weight of 50 and Length Weight of 3;

[0043] (f) a polynucleotide comprising at least 25 nucleotides in lengthwhich hybridizes under high stringency conditions to a polynucleotidehaving the sequence set forth in SEQ ID NO: 1, 7, 9, 11, 13, 15, 17, 19,or 21;

[0044] (g) a polynucleotide encoding the protein of SEQ ID NO: 2, 8, 10,12, 14, 16, 18, 20, or 22, wherein the polynucleotide is from a plantother than Arabidopsis;

[0045] (h) a polynucleotide having the sequence set forth in SEQ ID NO:1, 7, 9, 11, 13, 15, 17, 19, or 21; and

[0046] (i) a polynucleotide complementary to a polynucleotide of (a)through (h).

[0047] In many cases of apomixis maternal tissues such as the nucellusor inner integument “bud off” producing somatic embryos. These embryosthen develop normally into seed. Since meiosis and fertilization arecircumvented, the plants developing from such seed are geneticallyidentical to the maternal plant. Expression of the leafy cotyledon 1gene in the nucellus integument, or cell specific expression in themegaspore mother cell would trigger embryo formation from maternaltissues.

[0048] Producing a seed identical to the parent has many advantages. Forexample high yielding hybrids could be used in seed production tomultiply identical copies of high yielding hybrid seed. This wouldgreatly reduce seed cost as well as increase the number of genotypeswhich are commercially available. Genes can be evaluated directly incommercial hybrids since the progeny would not segregate. This wouldsave years of back crossing.

[0049] Apomixis would also provide a method of containment of transgeneswhen coupled with male sterility. The construction of male sterileautonomous agamospermy would prevent genetically engineered traits fromhybridizing with weedy relatives.

[0050] Gene stacking would be relatively easy with apomixis. Hybridscould be successively re-transformed with various new traits andpropagated via apomixis. The traits would not need to be linked sinceapomixis avoids the problems associated with segregation.

[0051] Apomixis can provide a reduction in gene silencing. Genesilencing is frequently seen following meiotic divisions. Since meioticdivisions never occur, it may be possible to eliminate or reduce thefrequency of gene silencing. Apomixis can also be used stabilizedesirable phenotypes with complex traits such as hybrid vigor. Suchtraits could easily be maintained and multiplied indefinitely viaapomixis.

[0052] The Cauliflower Mosaic Virus 35S promoter has been used tooverexpress LEC1 during Agrobacterium-mediated in planta transformationof Arabidopsis (Harada et al., WO 98/37184). As pointed out by Harada etal., 35S is a strong promoter, and in their experiments it was foundthat 35S:LEC1 did not improve transformation and actually appeared tohinder it (transformation efficiency with 35S:LEC1 was estimated to be0.6% of that obtained normally). Thus, overexpression in a cell typesuch as those in the gametophytic stage of development may beinappropriate and detrimental to the transformation process andsuccessful recovery of transformed progeny. In contrast, we have shownthat ectopic expression of the LEC1 gene under the appropriate controlelements (including tissue specific and/or inducible promoters) and inthe appropriate plant cells can be used to stimulate embryo formation intissues/genotypes normally not amenable to culture. Likewise ectopicexpression in genotypes amenable to culture can increase the number ofembryo precursor cells (or increase the number that develop intoembryos) leading to an increase in transformation frequency. Transientexpression using RNA or protein may be sufficient to initiate thecascade of events leading to embryo formation. This would be valuable insuch target tissues as maize scutella, immature leaf bases, immaturetassels, etc. The LEC1 gene could be used as a positive selectablemarker, i.e. triggering embryogenesis in transgenic cells withoutkilling the surrounding wild-type cells. This would happen since thecells receiving the introduced gene would undergo embryogenesis or intissues already undergoing embryogenesis LEC1 expression would stimulatemore rapid reiteration of somatic embryos.

[0053] It has been shown through sequence similarity that theArabidopsis LEC1 polypeptide is homologous to the HAP3 subunit of the“CCAAT-box binding factor” class of eukaryotic transcriptionalactivators (Lotan et al., 1998, Cell 93:1195-1205). This class ofproteins, which consist of Hap2/3/4 and 5, form a heteroligomerictranscriptional complex, that appears to activate specific gene sets ineukaryotes. Certain members of this family such as Hap2 and Hap5 appearto be ubiquitously expressed, while different Hap3 members are underdevelopmental or environmental regulation. Plant HAP3 polypeptides canbe recognized by a high degree of sequence identity to other HAP3homologs in the “B domain” of the protein. For example, the B domain forthe Arabidopsis LEC1, from amino acid residue 28 to residue 117, sharesbetween 55% and 63% identity (75-85% similarity) to other members of theHAP3 family, including maize (HAP3), chicken, lamprey, Xenopus, human,mouse, Emericella nidulens, Schizosaccharomyces pombe, Saccharomycescerevisiae and Kluuyveromyces lactis (Lotan et al., 1998).

[0054] Expression of the LEC1 gene in transformed cells initiates embryodevelopment and stimulates development of pre-existing embryos.Normally, LEC1 expression is necessary for proper embryo maturation inthe latter stages of embryo development, and LEC1 transgene expressionthus may also promote these processes. The combined effect of theseimpacts on somatic embryogenesis is not only to stimulate growth oftransformed cells, but also to insure that transformed somatic embryosdevelop in a normal, viable fashion (increasing the capacity oftransformed somatic embryos to germinate vigorously). Continued ectopicoverexpression beyond embryo maturation may negatively impactgermination and vegetative plant growth (which may necessitatedown-regulation of the LEC1 transgene during these stages ofdevelopment.

[0055] Expression of the LEC1 gene will stimulate growth in cells withthe potential to initiate or maintain embryogenic growth. Cells inestablished meristems or meristem-derive cell lineages may be less proneto undergo the transition to embryos. In addition, transformationmethods that target certain reproductive tissues (or cells) such asvacuum-infiltration of Agrobacterium into Arabidopsis may havedetrimental effects on recovery of transformants (triggering genesassociated with embryogenesis may disrupt the proper functioning ofthese cells).

[0056] The polypeptides encoded by the present plant LEC1 genes can bedistinguished from non-LEC HAP3 proteins by using the diagnostic motifshown in SEQ ID NO: 23.

[0057] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot or dicot. In preferred embodiments the monocot is corn,sorghum, barley, wheat, millet, or rice. Preferred dicots includesoybeans, sunflower, canola, alfalfa, potato, or cassava.

[0058] Functional fragments included in the invention can be obtainedusing primers which selectively hybridize under stringent conditions.Primers are generally at least 12 bases in length and can be as high as200 bases, but will generally be from 15 to 75, preferably from 15 to50. Functional fragments can be identified using a variety of techniquessuch as restriction analysis, Southern analysis, primer extensionanalysis, and DNA sequence analysis.

[0059] The present invention includes a plurality of polynucleotidesthat encode for the identical amino acid sequence. The degeneracy of thegenetic code allows for such “silent variations” which can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Additionally, the presentinvention includes isolated nucleic acids comprising allelic variants.The term “allele” as used herein refers to a related nucleic acid of thesame gene.

[0060] Variants of nucleic acids included in the invention can beobtained, for example, by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like. See, for example, Ausubel, pages 8.0.3- 8.5.9.Also, see generally, McPherson (ed.), DIRECTED MUTAGENESIS: A PracticalApproach, (IRL Press, 1991). Thus, the present invention alsoencompasses DNA molecules comprising nucleotide sequences that havesubstantial sequence similarity with the inventive sequences.

[0061] Variants included in the invention may contain individualsubstitutions, deletions or additions to the nucleic acid or polypeptidesequences which alters, adds or deletes a single amino acid or a smallpercentage of amino acids in the encoded sequence is a “conservativelymodified variant” where the alteration results in the substitution of anamino acid with a chemically similar amino acid. When the nucleic acidis prepared or altered synthetically, advantage can be taken of knowncodon preferences of the intended host.

[0062] The present invention also includes “shufflents” produced bysequence shuffling of the inventive polynucleotides to obtain a desiredcharacteristic. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA94:4504-4509 (1997).

[0063] The present invention also includes the use of 5′ and/or 3′ UTRregions for modulation of translation of heterologous coding sequences.Positive sequence motifs include translational initiation consensussequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res.13:7375 (1985)). Negative elements include stable intramolecular 5′ UTRstem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUGsequences or short open reading frames preceded by an appropriate AUG inthe 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284(1988)).

[0064] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency. Codonusage in the coding regions of the polynucleotides of the presentinvention can be analyzed statistically using commercially availablesoftware packages such as “Codon Preference” available from theUniversity of Wisconsin Genetics Computer Group (see Devereaux et al.,Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman KodakCo., New Haven, Conn.).

[0065] For example, the inventive nucleic acids can be optimized forenhanced expression in plants of interest. See, for example, EPA0359472;WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Inthis manner, the polynucleotides can be synthesized utilizingplant-preferred codons. See, for example, Murray et al. (1989) NucleicAcids Res. 17:477-498, the disclosure of which is incorporated herein byreference.

[0066] The present invention provides subsequences comprising isolatednucleic acids containing at least 16 contiguous bases of the inventivesequences. For example the isolated nucleic acid includes thosecomprising at least 16, 20, 25, 30, 40, 50, 60, 75 or 100 contiguousnucleotides of the inventive sequences. Subsequences of the isolatednucleic acid can be used to modulate or detect gene expression byintroducing into the subsequences compounds which bind, intercalate,cleave and/or crosslink to nucleic acids.

[0067] The nucleic acids of the invention may conveniently comprise amulti-cloning site comprising one or more endonuclease restriction sitesinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention.

[0068] A polynucleotide of the present invention can be attached to avector, adapter, promoter, transit peptide or linker for cloning and/orexpression of a polynucleotide of the present invention. Additionalsequences may be added to such cloning and/or expression sequences tooptimize their function in cloning and/or expression, to aid inisolation of the polynucleotide, or to improve the introduction of thepolynucleotide into a cell. Use of cloning vectors, expression vectors,adapters, and linkers is well known and extensively described in theart. For a description of such nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla,Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

[0069] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library.

[0070] Exemplary total RNA and mRNA isolation protocols are described inPlant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997); and, Current Protocols in MolecularBiology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995). Total RNA and mRNA isolation kits are commerciallyavailable from vendors such as Stratagene (La Jolla, Calif.), Clonetech(Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli,Pa.). See also, U.S. Pat. Nos. 5,614,391 and 5,459,253.

[0071] Typical cDNA synthesis protocols are well known to the skilledartisan and are described in such standard references as: PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel etal., Eds., Greene Publishing and Wiley-Interscience, New York (1995).cDNA synthesis kits are available from a variety of commercial vendorssuch as Stratagene or Pharmacia.

[0072] An exemplary method of constructing a greater than 95% purefull-length cDNA library is described by Carninci et al., Genomics,37:327-336 (1996). Other methods for producing full-length libraries areknown in the art. See, e.g., Edery et al., Mol. CellBiol.15(6):3363-3371 (1995); and PCT Application WO 96/34981.

[0073] It is often convenient to normalize a cDNA library to create alibrary in which each clone is more equally represented. A number ofapproaches to normalize cDNA libraries are known in the art.Construction of normalized libraries is described in Ko, Nucl. Acids.Res. 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soareset al., Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

[0074] Subtracted cDNA libraries are another means to increase theproportion of less abundant cDNA species. See, Foote et al. in, PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); Kho and Zarbl, Technique 3(2):58-63 (1991); Sive and St.John, Nucl. Acids Res. 16(22):10937 (1988); Current Protocols inMolecular Biology, Ausubel et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); and, Swaroop et al., Nucl. AcidsRes. 19(8):1954 (1991). cDNA subtraction kits are commerciallyavailable. See, e.g., PCR-Select (Clontech).

[0075] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel et al., Eds., Greene Publishing and Wiley-Interscience, New York(1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

[0076] The cDNA or genomic library can be screened using a probe basedupon the sequence of a nucleic acid of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous polynucleotides in the same ordifferent plant species. Those of skill in the art will appreciate thatvarious degrees of stringency of hybridization can be employed in theassay; and either the hybridization or the wash medium can be stringent.The degree of stringency can be controlled by temperature, ionicstrength, pH and the presence of a partially denaturing solvent such asformamide.

[0077] Typically, stringent hybridization conditions will be those inwhich the salt concentration is less than about 1.5 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

[0078] For purposes of defining the invention the following conditionsare provided. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. Typically the time ofhybridization is from 4 to 16 hours.

[0079] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

[0080] The nucleic acids of the invention can be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related polynucleotidesdirectly from genomic DNA or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes.

[0081] Examples of techniques useful for in vitro amplification methodsare found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methodsand Applications, Innis et al., Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used toimprove yield of long PCR products. PCR-based screening methods havealso been described. Wilfinger et al. describe a PCR-based method inwhich the longest cDNA is identified in the first step so thatincomplete clones can be eliminated from study. BioTechniques22(3):481-486 (1997).

[0082] In one aspect of the invention, nucleic acids can be amplifiedfrom a plant nucleic acid library. The nucleic acid library may be acDNA library, a genomic library, or a library generally constructed fromnuclear transcripts at any stage of intron processing. Libraries can bemade from a variety of plant tissues. Good results have been obtainedusing mitotically active tissues such as shoot meristems, shoot meristemcultures, embryos, callus and suspension cultures, immature ears andtassels, and young seedlings. The cDNAs of the present invention wereobtained from immature zygotic embryo and regenerating callus libraries.

[0083] Alternatively, the sequences of the invention can be used toisolate corresponding sequences in other organisms, particularly otherplants, more particularly, other monocots. In this manner, methods suchas PCR, hybridization, and the like can be used to identify suchsequences having substantial sequence similarity to the sequences of theinvention. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). and Innis et al. (1990), PCR Protocols: A Guide toMethods and Applications (Academic Press, New York). Coding sequencesisolated based on their sequence identity to the entire inventive codingsequences set forth herein or to fragments thereof are encompassed bythe present invention.

[0084] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99 (1979);the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triestermethod described by Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168(1984); and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0085] The nucleic acids of the present invention include thoseamplified using the following primer pairs: SEQ ID NOS: 3-6 and 9-12.

Expression Cassettes

[0086] In another embodiment expression cassettes comprising isolatednucleic acids of the present invention are provided. An expressioncassette will typically comprise a polynucleotide of the presentinvention operably linked to transcriptional initiation regulatorysequences which will direct the transcription of the polynucleotide inthe intended host cell, such as tissues of a transformed plant.

[0087] The construction of such expression cassettes which can beemployed in conjunction with the present invention is well known tothose of skill in the art in light of the present disclosure. See, e.g.,Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold SpringHarbor, N.Y.; (1989); Gelvin et al.; Plant Molecular Biology Manual(1990); Plant Biotechnology: Commercial Prospects and Problems, eds.Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993);and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts;CRC Press, Inc., USA; (1992); each incorporated herein in its entiretyby reference.

[0088] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible, constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0089] Constitutive, tissue-preferred or inducible promoters can beemployed. Examples of constitutive promoters include the cauliflowermosaic virus (CaMV) 35S transcription initiation region, the 1′- or2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the actinpromoter, the ubiquitin promoter, the histone H2B promoter (Nakayama etal., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter,and other transcription initiation regions from various plant genesknown in the art.

[0090] Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, the PPDK promoter which is inducible by light,the In2 promoter which is safener induced, the ERE promoter which isestrogen induced and the Pepcarboxylase promoter which is light induced.

[0091] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruit, seeds, or flowers. An exemplary promoteris the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051). Examples of seed-preferred promoters include, but are notlimited to, 27 kD gamma zein promoter and waxy promoter, Boronat, A.,Martinez, M. C., Reina, M., Puigdomenech, P. and Palau, J.; Isolationand sequencing of a 28 kD glutelin-2 gene from maize: Common elements inthe 5′ flanking regions among zein and glutelin genes; Plant Sci.47:95-102 (1986) and Reina, M., Ponte, I., Guillen, P., Boronat, A. andPalau, J., Sequence analysis of a genomic clone encoding a Zc2 proteinfrom Zea mays W64 A, Nucleic Acids Res. 18(21):6426 (1990). See thefollowing site relating to the waxy promoter: Kloesgen, R. B., Gierl,A., Schwarz-Sommer, Z. S. and Saedler, H., Molecular analysis of thewaxy locus of Zea mays, Mol. Gen. Genet. 203:237-244 (1986). Thedisclosures each of these are incorporated herein by reference in theirentirety.

[0092] Preferably a weak constitutive promoter, such as the Nospromoter, an inducible promoter, such as In2, or a nucellus-preferred orintegument-preferred promoter are used to induce apospory. For examplethe barley or maize Nuc1 promoter, the maize Cim 1 promoter or the maizeLTP2 promoter can be used to preferentially express in the nucellus. Seefor example U.S. Ser. No. 60/097,233 filed Aug. 20, 1998 the disclosureof which is incorporated herein by reference.

[0093] Either heterologous or non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue.

[0094] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0095] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0096] The vector comprising the sequences from a polynucleotide of thepresent invention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic or herbicide resistance. Suitable genes includethose coding for resistance to the antibiotics spectinomycin andstreptomycin (e.g., the aada gene), the streptomycin phosphotransferase(SPT) gene coding for streptomycin resistance, the neomycinphosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance, the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance.

[0097] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to inhibitaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron. While useful in conjunctionwith the above antibiotic and herbicide-resistance selective markers(i.e. use of the LEC1 gene can increase transformation frequencies whenusing chemical selection), a prefered use of LEC1 expression takesadvantage of this gene conferring a growth advantage to transformedcells without the need for inhibitory compounds to retardnon-transformed growth. Thus, LEC1 transformants are recovered basedsolely on their differential growth advantage.

[0098] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol. 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene, 61:1-11 (1987) and Berger et al., Proc. Natl.Acad. Sci. USA 86:8402-8406 (1989). Another useful vector herein isplasmid pBI101.2 that is available from Clontech Laboratories, Inc.(Palo Alto, Calif.).

[0099] A variety of plant viruses that can be employed as vectors areknown in the art and include cauliflower mosaic virus (CaMV),geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0100] A polynucleotide of the present invention can be expressed ineither sense or anti-sense orientation as desired. In plant cells, ithas been shown that antisense RNA inhibits gene expression by preventingthe accumulation of mRNA which encodes the enzyme of interest, see,e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

[0101] Another method of suppression is sense suppression. Introductionof nucleic acid configured in the sense orientation has been shown to bean effective means by which to block the transcription of target genes.For an example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323.

[0102] Recent work has shown suppression with the use of double strandedRNA. Such work is described in Tabara et al., Science 282:5388:430-431(1998).

[0103] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

[0104] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R.B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home, et al., J. Am. Chem. Soc. (1990) 112:2435-2437.Use of N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been described by Webb andMatteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res(1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and, 5,681,941.

Proteins

[0105] In another aspect, the invention relates to an isolated proteincomprising a member selected from the group consisting of:

[0106] (a) a polypeptide comprising at least 25 contiguous amino acidsof SEQ ID NO: 2, 8, 10, 12, 14, 16, 18, 20, or 22;

[0107] (b) a polypeptide which is a plant HAP3-type CCATT-box bindingtranscriptional activator that regulates gene expression during embryodevelopment and maturation;

[0108] (c) a polypeptide comprising at least 60% sequence identity toSEQ ID NO: 2, 12, 14, 16, 20, or 22, or 70% sequence identity to SEQ IDNO: 8,10, or 18, wherein the % sequence identity is based on the entiresequence and is determined by GAP analysis using Gap Weight of 12 andLength Weight of 4;

[0109] (d) a polypeptide encoded by a nucleic acid of claim 1;

[0110] (e) a polypeptide encoded by a nucleic acid of SEQ ID NO: 1, 7,9, 11, 13, 15, 17, 19, or 21; and

[0111] (f) a polypeptide having the sequence set forth in SEQ ID NO: 2,8, 10, 12, 14, 16, 18, 20, or 22.

[0112] Proteins of the present invention include proteins derived fromthe native protein by deletion (so-called truncation), addition orsubstitution of one or more amino acids at one or more sites in thenative protein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Methods for such manipulationsare generally known in the art.

[0113] For example, amino acid sequence variants of the polypeptide canbe prepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

[0114] In constructing variants of the proteins of interest,modifications to the nucleotide sequences encoding the variants will bemade such that variants continue to possess the desired activity.Obviously, any mutations made in the DNA encoding the variant proteinmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. See EP Patent Application Publication No. 75,444.

[0115] The isolated proteins of the present invention include apolypeptide comprising at least 23 contiguous amino acids encoded by anyone of the nucleic acids of the present invention, or polypeptides whichare conservatively modified variants thereof. The proteins of thepresent invention or variants thereof can comprise any number ofcontiguous amino acid residues from a polypeptide of the presentinvention, wherein that number is selected from the group of integersconsisting of from 23 to the number of residues in a full-lengthpolypeptide of the present invention. Optionally, this subsequence ofcontiguous amino acids is at least 25, 30, 35, or 40 amino acids inlength, often at least 50, 60, 70, 80, or 90 amino acids in length.

[0116] The present invention includes catalytically active polypeptides(i.e., enzymes). Catalytically active polypeptides will generally have aspecific activity of at least 20%, 30%, or 40%, and preferably at least50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% that ofthe native (non-synthetic), endogenous polypeptide. Further, thesubstrate specificity (k_(cat)/K_(m)) is optionally substantiallysimilar to the native (non-synthetic), endogenous polypeptide.Typically, the K_(m) will be at least 30%, 40%, or 50%, that of thenative (non-synthetic), endogenous polypeptide; and more preferably atleast 60%, 70%, 80%, or 90%. Methods of assaying and quantifyingmeasures of enzymatic activity and substrate specificity(k_(cat)/K_(m)), are well known to those of skill in the art.

[0117] The present invention includes modifications that can be made toan inventive protein. In particular, it may be desirable to diminish theactivity of the LEC1 gene. Other modifications may be made to facilitatethe cloning, expression, or incorporation of the targeting molecule intoa fusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,poly His) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

[0118] Using the nucleic acids of the present invention, one may expressa protein of the present invention in a recombinantly engineered cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The cells produce the protein in a non-natural condition (e.g., inquantity, composition, location, and/or time), because they have beengenetically altered through human intervention to do so.

[0119] Typically, an intermediate host cell will be used in the practiceof this invention to increase the copy number of the cloning vector.With an increased copy number, the vector containing the gene ofinterest can be isolated in significant quantities for introduction intothe desired plant cells.

[0120] Host cells that can be used in the practice of this inventioninclude prokaryotes, including bacterial hosts such as Eschericia coli,Salmonella typhimurium, and Serratia marcescens. Eukaryotic hosts suchas yeast or filamentous fungi may also be used in this invention. Sincethese hosts are also microorganisms, it will be essential to ensure thatplant promoters which do not cause expression of the polypeptide inbacteria are used in the vector.

[0121] Commonly used prokaryotic control sequences include such commonlyused promoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0122] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva, et al., Gene22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)).

[0123] Synthesis of heterologous proteins in yeast is well known. SeeSherman, F., et al., Methods in Yeast Genetics, Cold Spring HarborLaboratory (1982). Two widely utilized yeast for production ofeukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

[0124] A protein of the present invention, once expressed, can beisolated from yeast by lysing the cells and applying standard proteinisolation techniques to the lysates. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay of other standard immunoassay techniques.

[0125] The proteins of the present invention can also be constructedusing non-cellular synthetic methods. Solid phase synthesis of proteinsof less than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield, et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicycylohexylcarbodiimide)) is known to those of skill.

[0126] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. Detection of the expressed proteinis achieved by methods known in the art and include, for example,radioimmunoassays, Western blotting techniques or immunoprecipitation.

[0127] The present invention further provides a method for modulating(i.e., increasing or decreasing) the concentration or composition of thepolypeptides of the present invention in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the composition (i.e., the ratio of the polypeptides of thepresent invention) in a plant.

[0128] The method comprises transforming a plant cell with an expressioncassette comprising a polynucleotide of the present invention to obtaina transformed plant cell, growing the transformed plant cell underconditions allowing expression of the polynucleotide in the plant cellin an amount sufficient to modulate concentration and/or composition inthe plant cell.

[0129] In some embodiments, the content and/or composition ofpolypeptides of the present invention in a plant may be modulated byaltering, in vivo or in vitro, the promoter of a non-isolated gene ofthe present invention to up- or down-regulate gene expression. In someembodiments, the coding regions of native genes of the present inventioncan be altered via substitution, addition, insertion, or deletion todecrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulationof the protein involves using PEST sequences that provide a target fordegradation of the protein. It has been observed that high levels ofLEC1 prevent germination. See Lotan et al., Cell 1998 Jun. 26;93(7):1195-1205. Thus, temporal regulation of LEC1 expression may bedesirable in certain species to permit proper germination, vegetativegrowth, flowering and reproduction.

[0130] In some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart.

[0131] In general, concentration or composition is increased ordecreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%relative to a native control plant, plant part, or cell lacking theaforementioned expression cassette. Modulation in the present inventionmay occur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds which activate expression from these promoters arewell known in the art. In preferred embodiments, the polypeptides of thepresent invention are modulated in monocots or dicots, preferably maize,soybeans, sunflower, sorghum, canola, wheat, alfalfa, rice, barley andmillet.

[0132] Means of detecting the proteins of the present invention are notcritical aspects of the present invention. In a preferred embodiment,the proteins are detected and/or quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology, Vol. 37:Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds.(1991). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, e.g., those reviewed inEnzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980);Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers B.V.,Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A PracticalGuide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles andPractice of Immunoassays, Price and Newman Eds., Stockton Press, NY(1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY(1988).

[0133] Typical methods include Western blot (immunoblot) analysis,analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

[0134] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0135] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

[0136] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0137] The proteins of the present invention can be used for identifyingcompounds that bind to (e.g., substrates), and/or increase or decrease(i.e., modulate) the enzymatic activity of, catalytically activepolypeptides of the present invention. The method comprises contacting apolypeptide of the present invention with a compound whose ability tobind to or modulate enzyme activity is to be determined. The polypeptideemployed will have at least 20%, preferably at least 30% or 40%, morepreferably at least 50% or 60%, and most preferably at least 70% or 80%of the specific activity of the native, full-length polypeptide of thepresent invention (e.g., enzyme). Methods of measuring enzyme kineticsare well known in the art. See, e.g., Segel, Biochemical Calculations,2^(nd) ed., John Wiley and Sons, New York (1976).

[0138] Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies are known to persons of skill.

[0139] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Basic and Clinical Immunology,4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding,Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press,New York, N.Y. (1986); and Kohler and Milstein, Nature 256:495-497(1975).

[0140] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotechnology, 14:309-314 (1996)).Alternatively, high avidity human monoclonal antibodies can be obtainedfrom transgenic mice comprising fragments of the unrearranged humanheavy and light chain lg loci (i.e., minilocus transgenic mice).Fishwild et al., Nature Biotech., 14:845-851 (1996). Also, recombinantimmunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567;and Queen et al., Proc. Natl. Acad. Sci. 86:10029-10033 (1989).

[0141] The antibodies of this invention can be used for affinitychromatography in isolating proteins of the present invention, forscreening expression libraries for particular expression products suchas normal or abnormal protein or for raising anti-idiotypic antibodieswhich are useful for detecting or diagnosing various pathologicalconditions related to the presence of the respective antigens.

[0142] Frequently, the proteins and antibodies of the present inventionwill be labeled by joining, either covalently or non-covalently, asubstance which provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

Transfection/Transformation of Cells

[0143] The method of transformation/transfection is not critical to theinstant invention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod which provides for efficient transformation/transfection may beemployed.

[0144] A DNA sequence coding for the desired polynucleotide of thepresent invention, for example a cDNA or a genomic sequence encoding afull length protein, can be used to construct an expression cassettewhich can be introduced into the desired plant. Isolated nucleic acidacids of the present invention can be introduced into plants accordingtechniques known in the art. Generally, expression cassettes asdescribed above and suitable for transformation of plant cells areprepared.

[0145] Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical, scientific, andpatent literature. See, for example, Weising et al., Ann. Rev. Genet.22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG poration, particle bombardment, silicon fiberdelivery, or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp.197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G. C.Phillips. Springer-Verlag Berlin Heidelberg New York, 1995.Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0146] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327:70-73 (1987).

[0147]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maizeis described in WO 98/32326. Agrobacterium transformation of soybean isdescribed in U.S. Pat. No. 5,563,055.

[0148] Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, Vol. 6, PWJ Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J,.In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A. rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, (1984)),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA87:1228, (1990)).

[0149] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingpolynucleotides can be obtained by injection of the DNA intoreproductive organs of a plant as described by Pena et al., Nature,325:274 (1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as describedby Neuhaus et al., Theor. Appl. Genet., 75:30 (1987); and Benbrook etal., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp.27-54 (1986).

[0150] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

[0151] Altering the Culture Medium to Suppress Somatic Embryogenesis inNon-transformed Plant Cells and/or Tissues to Provide for a PositiveSection Means of Transformed Plant Cells

[0152] Using the following methods for controlling somaticembryogenesis, it is possible to alter plant tissue culture mediacomponents to suppress somatic embryogenesis in a plant species ofinterest (often having multiple components that potentially could beadjusted to impart this effect). Such conditions would not impart anegative or toxic in vitro environment for wild-type tissue, but insteadwould simply not produce a somatic embryogenic growth form. Introducinga transgene such as LEC1 will stimulate somatic embryogenesis and growthin the transformed cells or tissue, providing a clear differentialgrowth screen useful for identifying transformants.

[0153] Altering a wide variety of media components can modulate somaticembryogenesis (either stimulating or suppressing embryogenesis dependingon the species and particular media component). Examples of mediacomponents which, when altered, can stimulate or suppress somaticembryogenesis include;

[0154] 1) the basal medium itself (macronutrient, micronutrients andvitamins; see T. A. Thorpe, 1981 for review, “Plant Tissue Culture:Methods and Applications in Agriculture”, Academic Press, NY),

[0155] 2) plant phytohormones such as auxins (indole acetic acid, indolebutyric acid, 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid,picloram, dicamba and other functional analogues), cytokinins (zeatin,kinetin, benzyl amino purine, 2-isopentyl adenine andfunctionally-related compounds) abscisic acid, adenine, and gibberellicacid,

[0156] 3) and other compounds that exert “growth regulator” effects suchas coconut water, casein hydrolysate, and proline, and

[0157] 4) the type and concentration of gelling agent, pH and sucroseconcentration.

[0158] Changes in the individual components listed above (or in somecases combinations of components) have been demonstrated in theliterature to modulate in vitro somatic embryogenesis across a widerange of dicotyledonous and monocotyledonous species. For a compilationof examples, see E. F. George et al. 1987. Plant Tissue Culture Media.Vol. 1: Formulations and Uses. Exergetics, Ltd., Publ., Edington,England.

Transgenic Plant Regeneration

[0159] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell, 2:603-618 (1990).

[0160] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

[0161] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0162] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). For maize cell culture andregeneration see generally, The Maize Handbook, Freeling and Walbot,Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3^(rd) edition,Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis.(1988).

[0163] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed.

[0164] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings, via production of apomictic seed,or by tissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype.

[0165] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

[0166] Transgenic plants expressing a selectable marker can be screenedfor transmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then analyzed for protein expression by Western immunoblot analysisusing the specifically reactive antibodies of the present invention. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

[0167] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated. Alternatively, propagation of heterozygous transgenicplants could be accomplished through apomixis.

[0168] The present invention provides a method of genotyping a plantcomprising a polynucleotide of the present invention. Genotypingprovides a means of distinguishing homologs of a chromosome pair and canbe used to differentiate segregants in a plant population. Molecularmarker methods can be used for phylogenetic studies, characterizinggenetic relationships among crop varieties, identifying crosses orsomatic hybrids, localizing chromosomal segments affecting monogenictraits, map based cloning, and the study of quantitative inheritance.See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7,Clark, Ed., Springer-Verlag, Berlin (1997). For molecular markermethods, see generally, The DNA Revolution by Andrew H. Paterson 1996(Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) byAcademic Press/R. G. Landis Company, Austin, Tex., pp.7-21.

[0169] The particular method of genotyping in the present invention mayemploy any number of molecular marker analytic techniques such as, butnot limited to, restriction fragment length polymorphisms (RFLPs). RFLPsare the product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. Thus, the present inventionfurther provides a means to follow segregation of a gene or nucleic acidof the present invention as well as chromosomal sequences geneticallylinked to these genes or nucleic acids using such techniques as RFLPanalysis.

[0170] Plants which can be used in the method of the invention includemonocotyledonous and dicotyledonous plants. Preferred plants includemaize, wheat, rice, barley, oats, sorghum, millet, rye, soybean,sunflower, alfalfa, canola and cotton.

[0171] Seeds derived from plants regenerated from transformed plantcells, plant parts or plant tissues, or progeny derived from theregenerated transformed plants, may be used directly as feed or food, orfurther processing may occur.

[0172] All publications cited in this application are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

[0173] The present invention will be further described by reference tothe following detailed examples. It is understood, however, that thereare many extensions, variations, and modifications on the basic theme ofthe present invention beyond that shown in the examples and description,which are within the spirit and scope of the present invention.

EXAMPLES Example 1 Library Construction Used for the Maize LEC1 EST's

[0174] A. Total RNA Isolation

[0175] Total RNA was isolated from maize embryo and regenerating callustissues with TRIzol Reagent (Life Technology Inc. Gaithersburg, Md.)using a modification of the guanidine isothiocyanate/acid-phenolprocedure described by Chomczynski and Sacchi (Chomczynski, P., andSacchi, N. Anal. Biochem. 162, 156 (1987)). In brief, plant tissuesamples were pulverized in liquid nitrogen before the addition of theTRIzol Reagent, and then were further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation was conductedfor separation of an aqueous phase and an organic phase. The total RNAwas recovered by precipitation with isopropyl alcohol from the aqueousphase.

[0176] B. Poly(A)+ RNA Isolation

[0177] The selection of poly(A)+ RNA from total RNA was performed usingPolyATact system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringent condition and eluted by RNase-free deionizedwater.

[0178] C. cDNA Library Construction

[0179] cDNA synthesis was performed and unidirectional cDNA librarieswere constructed using the SuperScript Plasmid System (Life TechnologyInc. Gaithersburg, Md.). The first stand of cDNA was synthesized bypriming an oligo(dT) primer containing a Not I site. The reaction wascatalyzed by SuperScript Reverse Transcriptase II at 45° C. The secondstrand of cDNA was labeled with alpha-³²P-dCTP and a portion of thereaction was analyzed by agarose gel electrophoresis to determine cDNAsizes. cDNA molecules smaller than 500 base pairs and unligated adapterswere removed by Sephacryl-S400 chromatography. The selected cDNAmolecules were ligated into pSPORT1 vector in between of Not I and Sal Isites.

Example 2 Sequencing and cDNA Subtraction Procedures Used for Maize LEC1EST's

[0180] A. Sequencing Template Preparation

[0181] Individual colonies were picked and DNA was prepared either byPCR with M13 forward primers and M13 reverse primers, or by plasmidisolation. All the cDNA clones were sequenced using M13 reverse primers.

[0182] B. Q-bot Subtraction Procedure

[0183] cDNA libraries subjected to the subtraction procedure were platedout on 22×22 cm² agar plate at density of about 3,000 colonies perplate. The plates were incubated in a 37° C. incubator for 12-24 hours.Colonies were picked into 384-well plates by a robot colony picker,Q-bot (GENETIX Limited). These plates were incubated overnight at 37° C.

[0184] Once sufficient colonies were picked, they were pinned onto 22×22cm² nylon membranes using Q-bot. Each membrane contained 9,216 coloniesor 36,864 colonies. These membranes were placed onto agar plate withappropriate antibiotic. The plates were incubated at 37° C. forovernight.

[0185] After colonies were recovered on the second day, these filterswere placed on filter paper prewetted with denaturing solution for fourminutes, then were incubated on top of a boiling water bath foradditional four minutes. The filters were then placed on filter paperprewetted with neutralizing solution for four minutes. After excesssolution was removed by placing the filters on dry filter papers for oneminute, the colony side of the filters were place into Proteinase Ksolution, incubated at 37° C. for 40-50 minutes. The filters were placedon dry filter papers to dry overnight. DNA was then cross-linked tonylon membrane by UV light treatment.

[0186] Colony hybridization was conducted as described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A laboratoryManual, 2^(nd) Edition). The following probes were used in colonyhybridization:

[0187] 1. First strand cDNA from the same tissue from which the librarywas made to remove the most redundant clones.

[0188] 2. 48-192 most redundant cDNA clones from the same library basedon previous sequencing data.

[0189] 3. 192 most redundant cDNA clones in the entire corn sequencedatabase.

[0190] 4. A Sal-A20 oligo nucleotide: TCG ACC CAC GCG TCC GAA AAA AAAAAA AAA AAA AAA, removes clones containing a poly A tail but no cDNA.

[0191] 5. cDNA clones derived from rRNA.

[0192] The image of the autoradiography was scanned into computer andthe signal intensity and cold colony addresses of each colony wasanalyzed. Re-arraying of cold-colonies from 384 well plates to 96 wellplates was conducted using Q-bot.

Example 3 Identification of Maize LEC1 EST's from a Computer HomologySearch

[0193] Gene identities were determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol.215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches underdefault parameters for similarity to sequences contained in the BLAST“nr” database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences were analyzedfor similarity to all publicly available DNA sequences contained in the“nr” database using the BLASTN algorithm. The DNA sequences weretranslated in all reading frames and compared for similarity to allpublicly available protein sequences contained in the “nr” databaseusing the BLASTX algorithm (Gish, W. and States, D. J. (1993) NatureGenetics 3:266-272) provided by the NCBI. In some cases, the sequencingdata from two or more clones containing overlapping segments of DNA wereused to construct contiguous DNA sequences.

Example 4 Composition of cDNA Libraries Used to Isolate and SequenceAdditional cDNA Clones

[0194] cDNA libraries representing mRNAs from various corn, poppy,soybean and Vemonia tissues were prepared (see Table 1). Thecharacteristics of the libraries are described below. TABLE 1 cDNALibraries from Corn, Poppy, Soybean and Vernonia Tissue Corn endosperm20 days After pollination* Prickly poppy developing seeds Soybeanembryo, 6 to 10 days after flowering Soybean embryo, 13 days afterflowering Soybean embryogenic suspension 2 weeks after subcultureSoybean mature embryo 8 weeks after subculture Soybean embryogenicsuspension Vernonia developing seed*

[0195] cDNA libraries were prepared in Uni-ZAP™ XR vectors according tothe manufacturer's protocol (Stratagene Cloning Systems, La Jolla,Calif.). Conversion of the Uni-ZAP™ XR libraries into plasmid librarieswas accomplished according to the protocol provided by Stratagene. Uponconversion, cDNA inserts were contained in the plasmid vectorpBluescript. cDNA inserts from randomly picked bacterial coloniescontaining recombinant pBluescript plasmids were amplified viapolymerase chain reaction using primers specific for vector sequencesflanking the inserted cDNA sequences or plasmid DNA was prepared fromcultured bacterial cells. Amplified insert DNAs or plasmid DNAs weresequenced in dye-primer sequencing reactions to generate partial cDNAsequences (expressed sequence tags or “ESTs”; see Adams, M. D. et al.,(1991) Science 252:1651). The resulting ESTs were analyzed using aPerkin Elmer Model 377 fluorescent sequencer.

Example 5 Identification of cDNA Clones Obtained from Tissue Describedin Table 1

[0196] ESTs encoding plant transcription factors were identified byconducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., etal., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nim.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the last major release of theSWISS-PROT protein sequence database, EMBL, and DDBJ databases). ThecDNA sequences obtained in Example 1 were analyzed for similarity to allpublicly available DNA sequences contained in the “nr” database usingthe BLASTN algorithm provided by the National Center for BiotechnologyInformation (NCBI). The DNA sequences were translated in all readingframes and compared for similarity to all publicly available proteinsequences contained in the “nr” database using the BLASTX algorithm(Gish, W. and States, D. J. (1993) Nature Genetics 3:266-272 andAltschul, Stephen F., et al. (1997) Nucleic Acids Res. 25:3389-3402)provided by the NCBI.

Example 6 Identification of Protein Motifs Diagnostic for LEC1 Genes

[0197] To determine the structural requirements for a LEC1 gene, HAP3homologs were identified in our EST database and aligned. By analyzingsequence homology amongst the plant HAP3 family of transcriptionalactivators these sequences were observed to fall into a least twodistinctive groups. All of the HAP3 sequences derived from seed orembryo specific libraries form a distinctive LEC1 group that suggests acommon evolutionary origin (confirmed by phylodentrograms). For examplewithin the “B domain” of all plant LEC1 sequences examined, a highlyconserved CCAAT-box binding motif has been found to contain thenon-variable residues methionine, proline, isoleucine, alanine,asparagine, valine, and isoleucine (MPIANVI). LEC1 genes are highlydivergent outside of the region spanning the DNA binding and subunitinteraction motifs. The low levels of homology between these genes makeit difficult to identify these based solely on a hybridization strategy.Using sequences from maize, soybean, wheat, prickly poppy, Vernonia, andArabidopsis a motif diagnostic for LEC1 genes was identified, thespecific amino acid substitutions for these species was clarified(FIG. 1) and the positions at which amino acid substitutions occurwithin the LEC1 group was determined (SEQ ID NO: 23). Using Blast, themotif in SEQ ID NO: 23 was used to correctly distinguish LEC1's fromother closely related plant HAP3 transcriptional activators.

Example 7 Transformation and Regeneration of Maize Callus

[0198] Immature maize embryos from greenhouse or field grown High typeII donor plants were bombarded with a plasmid containing apolynucleotide of the invention (LEC1). The LEC1 polynucleotide wasoperably linked to a constitutive promoter such as nos, or an induciblepromoter, such as In2, plus a plasmid containing the selectable markergene PAT (Wohlleben et al. (1988) Gene 70:25-37) that confers resistanceto the herbicide Bialaphos fused to the Green Fluorescence protein.Transformation was performed as follows.

[0199] The ears were surface sterilized in 50% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos were excised and placed embryo axis side down(scutellum side up), 25 embryos per plate. These were cultured on 560 Lmedium 4 days prior to bombardment in the dark. Medium 560 L is anN6-based medium containing Eriksson's vitamins, thiamine, sucrose,2,4-D, and silver nitrate. The day of bombardment, the embryos weretransferred to 560 Y medium for 4 hours and were arranged within the2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L withhigh sucrose concentration).

[0200] A plasmid vector comprising a polynucleotide of the inventionoperably linked to the selected promoter was constructed. This plasmidDNA plus plasmid DNA containing a PAT selectable marker was precipitatedonto 1.1 μm (average diameter) tungsten pellets using a CaCl₂precipitation procedure as follows: 100 μl prepared tungsten particles(0.6 mg) in water, 20 μl (2 μg) DNA in TrisEDTA buffer (1 μg total), 100μl 2.5 M CaC1₂, 40 μl 0.1 M spermidine.

[0201] Each reagent was added sequentially to the tungsten particlesuspension. The final mixture was sonicated briefly. After theprecipitation period, the tubes were centrifuged briefly, liquidremoved, washed with 500 ml 100% ethanol, and centrifuged again for 30seconds. Again the liquid was removed, and 60 μl 100% ethanol was addedto the final tungsten particle pellet. For particle gun bombardment, thetungsten/DNA particles were briefly sonicated and 5 μl spotted onto thecenter of each macrocarrier and allowed to dry about 2 minutes beforebombardment.

[0202] The sample plates were bombarded at a distance of 8 cm from thestopping screen to the tissue, using a Dupont biolistics helium particlegun. All samples received a single shot at 650 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

[0203] Four to 12 hours post bombardment, the embryos were moved to 560P(a low osmoticum callus initiation medium similar to 560L but with lowersilver nitrate), for 3-7 days, then transferred to 560R selectionmedium, an N6 based medium similar to 560P containing 3 mg/literBialaphos, and subcultured every 2 weeks. Multicellular GFP cellclusters became visible after two weeks and their numbers wereperiodically recorded. After approximately 10 weeks of selection,selection-resistant GFP positive callus clones were sampled for PCR andactivity of the polynucleotide of interest. Positive lines weretransferred to 288J medium, an MS-based medium with lower sucrose andhormone levels, to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos were transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets were transferred tomedium in tubes for 7-10 days until plantlets were well established.Plants were then transferred to inserts in flats (equivalent to 2.5″pot) containing potting soil and grown for 1 week in a growth chamber,subsequently grown an additional 1-2 weeks in the greenhouse, thentransferred to Classic™ 600 pots (1.6 gallon) and grown to maturity.Plants are monitored for expression of the polynucleotide of interest.

Example 8 Ectopic Expression of Maize LEC1 to Induce SomaticEmbryogenesis

[0204] Using the genotype High type II as an example, embryos wereisolated and cultured on 560L medium for 3-5 days. Four to twelve hoursbefore bombardment these embryos were transferred to high osmotic 560Ymedium. Expression cassettes containing the LEC1 cDNA were thenco-introduced into the scutella of these embryos along with anexpression cassette containing the Pat gene fused to the GreenFluorescent protein using methods described in Example 7. Embryos from asingle ear were divided evenly between treatments. Four to 12 hoursfollowing bombardment embryos were then transferred back to a lowosmoticum callus initiation medium (560P) and incubated in the dark at26° C. After 3-7 days of culture these embryos were moved to 560Rselection medium. Cultures were then transferred every two weeks untiltransformed colonies appear. Cultures were also examined microscopicallyfor GFP expression. LEC1 expression was expected to stimulate adventiveembryo formation. This was apparent when the cultures were compared tocontrols (transformed without the LEC1 cDNA or non-induced).

[0205] A. Ectopic Expression of the Maize LEC1 Polynucleotide in Tobaccois Sufficient to Induce Somatic Embryogenesis in Tobacco Leaves

[0206] A maize LEC1 polynucleotide was placed into an agrobacteriumexpression cassette driven by the maize safener-induced In2 promoter(this promoter is leaky and expresses at low levels without induction).Also between the left and right T-DNA borders was the bar gene driven by35S promoter and the Green Fluorescence Protein driven by the ubiquitinpromoter. A similar construct was made without the LEC1 polynucleotideto be used as a control. Tobacco leaf discs from variety SR1 wereco-cultured with Agrobacterium as described by Horsch et al (1985,Science 227:1229-1231) except selecting with bialaphos rather thankanamycin. Transformants were selected on medium containing 3 mg/lbialaphos. Of the numerous transformed shoot obtained, ectopic embryoswere visible on the leaves of a single LEC1 transformant. None werevisible on control plants. Although the frequency was low, ectopicsomatic embryo formation was also reported to be a rare event inArabidopsis LEC1 transformants (Lotan et al 1998).

[0207] B. Transformation Frequency was Improved by LEC1 Introduced UsingParticle-mediated DNA Delivery.

[0208] A series of expression cassettes were made to evaluate theeffects of LEC1 expression on maize transformation. The maize LEC1polynucleotide was placed under the control of the In2 promoter (weaklyinduced with the auxin levels used under normal culture conditions andstrongly-induced with safener), the barley NUC1 promoter (expressedstrongly in the nucellus), the Ubiquitin promoter (strongly expressedconstitutively), and the nos promoter (weakly expressed constitutively).A frame-shift version of the In2:LEC1 cassette was made along with anIn2:ZM-NF-YB (designated as In2:HAP3 henceforth) construct (The maize ZMNF-YB is non-LEC1 type of HAP3 transcriptional activator (Li et alNucleic Acids Res. 20:1087-1091) for use as negative controls. All ofthese constructs were co-bombarded with the Pat˜GFP fusion construct(designated as PAT˜GFP) into high type II embryos as described inExample 7. Also, as in Example 7, immature embryos were harvested fromseparate ears, and the embryos from each ear were divided equallybetween treatments to account for ear-to-ear variability (for example,in an experiment comparing a control plasmid with this sameplasmid+LEC1, one-half the total embryos from each ear would be used foreach treatment. In some cases the control treatment contained thePat˜GFP construct co-bombarded with GUS. Transformation frequency wasdetermined by counting the numbers of embryos with large multicellularGFP-positive cells clusters using a GFP microscope, and representingthese as a percentage of the original number of embryos bombarded. Nodistinction was made between embryos with single or multiple events. Inall cases, the functional LEC1 expression cassettes increasedtransformation frequencies over the control treatment (the LEC1expression cassette also increased the incidence of multiple, i.e. 2-3,multicellular transgenic clones growing from the same immature embryo,but as stated above we only scored these as a single event, and areproviding a conservative representation of LEC1's ability to improvetransformation). For example, transformation frequencies in controltreatments for three consecutive experiments were 5.1, 7.4 and 0.8%. Inbalanced side-by-side comparisons for the same three experiments,transformation frequencies with the LEC1 polynucleotide(In2::LEC1::pinII) were 28.8, 25.7 and 12.4%, respectively. In additionto increasing the absolute number of transformants recovered from agiven amount of target tissue, LEC1 transformants appeared earlier thanthe control transformants (suggesting that the LEC1 polynucleotide alsostimulated growth rates).

[0209] As a more stringent control, an expression vector was constructedin which a LEC1 gene, frame-shifted immediately after the start codon,was placed behind the In2 promoter. In this experiment using embryosfrom 3 separate ears, the transformation frequencies in the control(frame-shifted LEC1) treatments were 2.7, 6.0 and 2.0%, while thetransformation frequencies for the LEC1 treatments were 62.7, 26 and42.7%. This demonstrated clearly that expression of the in-frame LEC1polynucleotide was associated with dramatic increases in transformationefficiency.

[0210] Increasing the promoter strength (driving LEC1 expression)increased transformation frequencies. For example, an experiment wasperformed to compare the In2, nos and UBI promoters. Based on ourexperience with these two promoters driving other genes, the In2promoter (in the absence of an inducer other than auxin from the medium)would drive expression at very low levels. The nos promoter has beenshown to drive moderately-low levels of transgene expression(approximately 10- to 30-fold lower than the maize ubiquitin promoter,but still stronger than In2 under the culture conditions used in thisexperiment). Two control treatments were used in this experiment; theframe-shifted LEC1 driven by the In2 promoter, or a maize In2:HAP3polynucleotide (a “non-LEC1 type” representative of the transcriptionalfactor family to which LEC1 belongs). Both control treatments resultedin low transformation frequencies. After 3 weeks, the transformationfrequency for the In2:frame-shift-LEC1 (FS) treatment was 4.8%, whilefor the In2:HAP3 treatment it was 2%. The In2:LEC1, nos:LEC1 andUBI:LEC1 treatments resulted in 14%, 28% and 30% transformationfrequencies, respectively. Within these treatments there was also anincrease in the overall frequency of large, rapidly growing calli. Forthe control treatments, the frequencies of large, vigorous GFP+ calli(relative to the starting number of embryos) was low (1.6 and 0% for theframe-shift or In2:HAP3, respectively). For the In2, nos and UBItreatments the overall frequencies of large, vigorous calli was 4, 13.3and 20%, respectively. This is consistent with the interpretation thatincreased LEC1 expression resulted in more rapid in vitro growth oftransgenic tissue. As is typical for transformation experiments scoredin this fashion, between 3-6 weeks the number of recovered transformantscontinues to rise. After 5 weeks (post-bombardment), the frequency ofhealthy, growing transformants was 4.8 and 7.3% for the FS and HAP3controls, while for the In2, nos and UBI-driven LEC1 treatments thefrequencies were 22, 29.3 and 35.3%.

[0211] C. Transformation Frequency was Improved by LEC1 Introduced UsingAgrobacterium.

[0212] The Agrobacterium strains containing the superbinary plasmidsdescribed in Example 8A were used to transformed High type II embryos.Briefly, colonies containing the engineered Agrobacterium were grown tolog phase in minimal A medium. Log phase cells were collected bycentrifugation and resuspended in 561Q medium (N6 salts, Eriksson'svitamins, 1.5 mg/l 2,4-D, 68.5 g/l sucrose, 36 g/l glucose, plus 20 mg/lacetosyringone). Immature embryos, 1.5-2 mm in length, were excised andimmersed in this solution at a concentration of 5×10⁸ bacterialcells/ml. Embryos were vortexed in this medium and allowed to sit for 5minutes. The embryos were then removed and placed on 562P medium (560Pmedium with 100 mM acetosyringone and incubated at 20° C. for 3 days.Embryos were moved again to 563N medium (an agar solidified mediumsimilar to 560P with 100 mg/l carbenicillin, 0.5 g/l MES and reduced2,4-D) and cultured at 28° C. for 3 days. Embryos were then moved to563O medium (563N medium with 3 mg/l bialaphos) and transferredthereafter every 14 days to fresh 563O medium.

[0213] Bialaphos resistant GFP+ colonies were counted using a GFPmicroscope and transformation frequencies were determined as describedin example 8B. Similar to particle gun experiments, transformationfrequencies were greatly increased in the LEC1 treatment. For example,transformation frequencies for the control treatment across embryostaken from 7 separate ears were 7.1, 40.9, 11.1, 7.4, 11.5, 12, 30.8,and 16.6%. The side-by-side comparison for the LEC treatment (in thesame order of ears as above) showed that transformation frequencies were13.5, 47, 55.8, 37.1, 40.6, 30, 57.1 and 40.8%. Averaged across all 7ears, the average transformation frequency for the control was 16.6%while that of the LEC1 treatment was 40.8%. This represents asubstantial increase for an already high baseline produced byAgrobacterium-mediated transformation. Comparing across ears, it wasobserved that the beneficial effects on transformation frequency werethe greatest when the control frequencies were low.

[0214] D. Transformants were Recovered Using LEC1 Expression UnderReduced Auxin Levels or in the Absence of Auxins in the Medium, and inthe Absence of Herbicide or Antibiotic Selection.

[0215] To determine if LEC1 could be used in a positive selectionscheme, particle gun transformation experiments were initiated asdescribed in Example 4 and transformants were selected on medium withnormal auxin levels, or on medium with reduced or no auxin, or visually(using GFP) on medium without bialaphos. Transformation frequencies werebased on the numbers of embryos with one or more multicellular GFPpositive cell clusters. In the first experiment to test this concept,there were two treatment variables. The first was that immature embryoswere bombarded with the control plasmid (UBI:PAT˜GFP) or withUBI:PAT˜GFP+In2:LEC1. The second variable was that the bombarded embryoswere divided onto either normal bialaphos-containing selection medium(with normal auxin levels of 2 mg/l 2,4-D), or medium with no bialaphosand reduced 2,4-D levels (0.5 mg/l). As expected from previous results,on bialaphos selection the LEC1 treatment resulted in a highertransformation frequency than the control (5.7 versus 2.5%). It was alsoanticipated that the low auxin medium (0.5 mg/l 2,4-D) would result inreduced growth rates. Consistent with this, for the control plasmidtreatment (UBI:PAT˜GFP), recovery of GFP-expressing (fluorescent)colonies was reduced relative to highly-effective bialaphos-selectiontreatment, dropping down to 0.6%. In contrast, it appeared that LEC1expression, through its stimulation of embryogenesis, may havecompensated for the low auxin environment, providing a growth advantageto the transgenic colonies, and maintaining the efficiency oftransformant recovery at 4.0% (still in the same range as theLEC1/bialaphos-selected treatment). It's clear from this result that theinclusion of LEC1 improved colony growth on reduced auxin relative tothe control.

[0216] On medium completely devoid of auxin, colonies were only observedin the LEC1 treatment. In this experiment, immature embryos werebombarded with either the control plasmid (UBI:PAT˜GFP) or withUBI:PAT˜GFP+In2:LEC1, and then plated either onto 3.0 mg/l bialaphos,2.0 mg/l 2,4-D medium or onto no-bialaphos, no 2,4-D medium (in thislatter treatment, wild-type maize callus will not exhibit embryogenicgrowth). Again, as expected, the LEC1 polynucleotide increasedtransformation to 22.7% over the control plasmid value of 8% on normalauxin-containing, bialaphos selection medium. Also, as expected, notransformants were recovered with the control plasmid on medium devoidof exogenous auxin. Surprisingly, in the LEC1 treated embryostransformants were recovered at a 4% frequency (this was still higherthan the control plasmid on bialaphos selection).

[0217] Even on auxin-containing medium, the LEC1 polynucleotide incombination with GFP+ expression can be used to recover transformantswithout chemical selection. For example, under these conditions therecovery of transformants was relatively efficient (16% compared to 18%for bialaphos selection), but this required more diligence than the low-or no-auxin treatments above to separate the GFP-expressing coloniesfrom the growing callus population.

[0218] E. LEC1 Improves the Embryogenic Phenotype and RegenerationCapacity of Inbreds.

[0219] Immature embryos from the inbred PHP38 were isolated, culturedand transformed as described in example 4 with the following changes.Embryos were initially cultured on 601H medium (a MS based medium with0.1 mg/l zeatin, 2 mg/l 2,4-D, MS and SH vitamins, proline, silvernitrate, extra potassium nitrate, casein hydrolysate, gelrite, 10 g/lglucose and 20 g/l sucrose). Prior to bombardment embryos were moved toa high osmoticum medium (modified Duncan's with 2 mg/l 2,4-D and 12%sucrose). Post bombardment, embryos were moved to 601H medium with 3mg/l bialaphos for two weeks. Embryos were then moved to 601H mediumwithout proline and casein hydrolysate with 3 mg/l bialaphos andtransferred every two weeks. Transformation frequency was determined bycounting the numbers of bialaphos resistant GFP-positive colonies.Colonies were also scored on whether they had an embryogenic(regenerable) or non-embryogenic phenotype. In PHP38, the LEC1polynucleotide increased transformation frequency and improved theregenerative potential of the callus. For example, a balanced experiment(the embryos from each harvested ear were divided equally betweentreatments) was conducted in which PHP38 immature embryos were bombardedwith the control plasmid (UBI::PAT˜GFP::pinII) in one treatment, withthe UBI::PAT˜GFP::pinII plasmid+In2::LEC1, or with theUBI::PAT˜GFP::pinII plasmid+nuc1::LEC1 (a maize nucellus-specificpromoter driving LEC1 expression). The frequency of GFP+ calli growingon bialaphos-containing media (relative to the starting number ofembryos) was determined 6 weeks after bombardment. For the controltreatment, the transformation frequency was 1.2%, while for the In2:LEC1and nuc1::LEC1 treatments the transformation frequencies were 3.2 and2.0% respectively. In addition, the presence of the LEC1 polynucleotideappeared to greatly improve the regeneration capacity of the recoveredtransformants. None of the control transformants (UBI::PAT˜GFP::pinIIalone) had an embryogenic, regenerable phenotype, while thetransformants recovered from the In2:LEC1 and nuc1::LEC1 treatments allexhibited a more vigorous, embryogenic growth pattern. This has beenborn out in the ability to recover plants. Callus from the In2:LEC1 andnuc1::LEC1 treatments has produced many healthy plants.

Example 9 Transient Expression of the LEC1 Polynucleotide Product toInduce Somatic Embryogenesis

[0220] It may be desirable to “kick start” somatic embryogenesis bytransiently expressing the LEC-1 polynucleotide product. This can bedone by delivering LEC1 5′capped polyadenylated RNA, expressioncassettes containing LEC-1 DNA, or LEC-1 protein. All of these moleculescan be delivered using a biolistics particle gun. For example 5′cappedpolyadenylated LEC1 RNA can easily be made in vitro using Ambion'smMessage mMachine kit. Following the procedure outline above RNA isco-delivered along with DNA containing an agronomically usefulexpression cassette. The cells receiving the RNA will immediately formsomatic embryos and a large portion of these will have integrated theagronomic gene. Plants regenerated from these embryos can then bescreened for the presence of the agronomic gene.

Example 10 Use of the Maize LEC1 to Induce Apomixis

[0221] Maize expression cassettes directing LEC1 expression to the innerintegument or nucellus can easily be constructed. An expression cassettedirecting expression of the LEC1 polynucleotide to the nucellus was madeusing the barley Nuc1 promoter. Embryos were co-bombarded with theselectable marker PAT fused to the GFP gene along with the nucellusspecific LEC1 expression cassette described above. Both inbred (PHP38)and GS3 transformants were obtained and regenerated as described inexamples 4 and 5. Transformation frequencies were also increased overthe control using the nuc1:LEC1 polynucleotide (see Example 8 above).

[0222] It is anticipated that the regenerated plants will then becapable of producing de novo embryos from LEC1 expressing nucellarcells. This is complemented by pollinating the ears to promote normalcentral cell fertilization and endosperm development. In anothervariation of this scheme, nuc1:LEC1 transformations could be done usinga FIE-null genetic background which would promote both de novo embryodevelopment and endosperm development without fertilization (see Ohad etal. 1999 The Plant Cell 11:407-415; also pending U.S. application Ser.No. 60/151575 filed Aug. 31, 1999). Upon microscopic examination of thedeveloping embryos it will be apparent that apomixis has occurred by thepresence of embryos budding off the nucellus. In yet another variationof this scheme the LEC1 polynucleotide could be delivered as describedabove into a homozygous zygotic-embryo-lethal genotype. Only theadventive embryos produced from somatic nucellus tissue would develop inthe seed.

Example 11 Expression of Chimeric Genes in Microbial Cells

[0223] The cDNAs encoding the instant transcription factors can beinserted into the T7 E. coli expression vector pBT430. This vector is aderivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) whichemploys the bacteriophage T7 RNA polymerase/T7 promoter system. PlasmidpBT430 was constructed by first destroying the EcoR I and Hind III sitesin pET-3a at their original positions. An oligonucleotide adaptorcontaining EcoR I and Hind III sites was inserted at the BamH I site ofpET-3a. This created pET-3aM with additional unique cloning sites forinsertion of genes into the expression vector. Then, the Nde I site atthe position of translation initiation was converted to an Nco I siteusing oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aMin this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0224] Plasmid DNA containing a cDNA may be appropriately digested torelease a nucleic acid fragment encoding the protein. This fragment maythen be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC).Buffer and agarose contain 10 μg/ml ethidium bromide for visualizationof the DNA fragment. The fragment can then be purified from the agarosegel by digestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe polynucleotide encoding the transcription factor are then screenedfor the correct orientation with respect to the T7 promoter byrestriction enzyme analysis.

[0225] For high level expression, a plasmid clone with the cDNA insertin the correct orientation relative to the T7 promoter can betransformed into E. coli strain BL21(DE3) (Studier et al. (1986) J. Mol.Biol. 189:113-130). Cultures are grown in LB medium containingampicillin (100 mg/L) at 25° C. At an optical density at 600 nm ofapproximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can beadded to a final concentration of 0.4 mM and incubation can be continuedfor 3 hours at 25° C. Cells are then harvested by centrifugation andre-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTTand 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glassbeads can be added and the mixture sonicated 3 times for about 5 secondseach time with a microprobe sonicator. The mixture is centrifuged andthe protein concentration of the supernatant determined. One μg ofprotein from the soluble fraction of the culture can be separated bySDS-polyacrylamide gel electrophoresis. Gels can be observed for proteinbands migrating at the expected molecular weight.

Example 12 Evaluating Compounds for Their Ability to Inhibit theActivity of Plant Transcription Factors

[0226] The transcription factors described herein may be produced usingany number of methods known to those skilled in the art. Such methodsinclude, but are not limited to, expression in bacteria as described inExample 6, or expression in eukaryotic cell culture, in planta, andusing viral expression systems in suitably infected organisms or celllines. The instant transcription factors may be expressed either asmature forms of the proteins as observed in vivo or as fusion proteinsby covalent attachment to a variety of enzymes, proteins or affinitytags. Common fusion protein partners include glutathione S-transferase(“GST”), thioredoxin (“Trx”), maltose binding protein, and C- and/orN-terminal hexahistidine polypeptide (“(His)₆”). The fusion proteins maybe engineered with a protease recognition site at the fusion point sothat fusion partners can be separated by protease digestion to yieldintact mature enzyme. Examples of such proteases include thrombin,enterokinase and factor Xa. However, any protease can be used whichspecifically cleaves the peptide connecting the fusion protein and theenzyme.

[0227] Purification of the instant transcription factors, if desired,may utilize any number of separation technologies familiar to thoseskilled in the art of protein purification. Examples of such methodsinclude, but are not limited to, homogenization, filtration,centrifugation, heat denaturation, ammonium sulfate precipitation,desalting, pH precipitation, ion exchange chromatography, hydrophobicinteraction chromatography and affinity chromatography, wherein theaffinity ligand represents a substrate, substrate analog or inhibitor.When the transcription factors are expressed as fusion proteins, thepurification protocol may include the use of an affinity resin which isspecific for the fusion protein tag attached to the expressed enzyme oran affinity resin containing ligands which are specific for the enzyme.For example, a transcription factor may be expressed as a fusion proteincoupled to the C-terminus of thioredoxin. In addition, a (His)₆ peptidemay be engineered into the N-terminus of the fused thioredoxin moiety toafford additional opportunities for affinity purification. Othersuitable affinity resins could be synthesized by linking the appropriateligands to any suitable resin such as Sepharose-4B. In an alternateembodiment, a thioredoxin fusion protein may be eluted usingdithiothreitol; however, elution may be accomplished using otherreagents which interact to displace the thioredoxin from the resin.These reagents include β-mercaptoethanol or other reduced thiol. Theeluted fusion protein may be subjected to further purification bytraditional means as stated above, if desired. Proteolytic cleavage ofthe thioredoxin fusion protein and the enzyme may be accomplished afterthe fusion protein is purified or while the protein is still bound tothe ThioBond™ affinity resin or other resin.

[0228] Crude, partially purified or purified enzyme, either alone or asa fusion protein, may be utilized in assays for the evaluation ofcompounds for their ability to inhibit enzymatic activition of thetranscription factors disclosed herein. Assays may be conducted underwell-known experimental conditions that permit optimal enzymaticactivity.

Example 13 LEC1 Expression Resulted in Increased Growth Rates, whichCould be Used as a Screening Criterion for Positive Selection ofTransformants

[0229] Using two promoters of increasing strength to drive LEC1expression in maize, it appeared that LEC1 stimulated callus growth overcontrol treatments and the stronger promoter driving LEC1 resulted infaster growth than with the low-level promoter. For example, anexperiment was performed to compare the In2 and nos promoters. As notedabove, based on our experience with these two promoters driving othergenes, the In2 promoter (in the absence of an inducer other than auxinfrom the medium) would drive expression at very low levels. The nospromoter has been shown to drive moderately-low levels of transgeneexpression (approximately 10- to 30-fold lower than the maize ubiquitinpromoter, but still stronger than In2 under the culture conditions usedin this experiment). One control treatment was used in this experiment,the UBI:PAT˜GFPmo:pinII construct by itself (with no LEC1). Hi-IIimmature embryos were bombarded as previously described, and transgenic,growing events were scored at 3 and 6 weeks. The control treatmentresulted in a transformation frequency of 0.8%. The In2:LEC1 andnos:LEC1 treatments resulted in transformation frequencies of 26.5 and40.7%, respectively.

[0230] Within these treatments there was also an increase in the overallfrequency of large, rapidly growing calli, relative to the controltreatment. For this data, the fresh weight of transformed calli wererecorded 2 months after bombardment. Assuming that all the transgenicevents started as single transformed cells within a few days afterbombardment, these weights represent the relative growth rate of thesetransformants during this period (all tissue was sub-cultured andweighed for each transformant; mean weights and standard deviations werecalculated for each treatment). For the control treatment, the meantransformant weight after two months was 37+/−15 mg (n=6). For theIn2:LEC1 and nos:LEC1 treatments, the mean transformant weights were126+/−106 and 441 +/−430 mg, respectively. If the control treatment wasset at a relative growth value of 1.0, this means that transformants inthe In2:LEC1 and nos:LEC1 treatments grew 3.4 and 12-fold faster thanthe control. From this data, it appears that increasing LEC1 expressionresulted in a concomitant increase in callus growth rate.

Example 14 The use of LEC1 Polynucleotide as a Positive Selection Systemfor Wheat Transformation and for Improving the Regeneration Capacity ofWheat Tissues

[0231] Method

[0232] Plant Material

[0233] Seeds of wheat Hybrinova lines NH535 and BO 014 were sown intosoil in plug trays for vernalisation at 6° C. for eight weeks.Vernalized seedlings were transferred in 8″ pots and grown in acontrolled environment room. The growth conditions used were; 1) soilcomposition: 75% L&P fine-grade peat, 12% screened sterilized loam, 10%6 mm screened, lime-free grit, 3% medium grade vermiculite, 3.5 kgOsmocote per m³ soil (slow-release fertiliser, 15-11-13 NPK plusmicronutrients), 0.5 kg PG mix per m³ (14-16-18 NPK granular fertiliserplus micronutrients, 2) 16 h photoperiod (400 W sodium lamps providingirradiance of ca. 750 μE s⁻¹ m⁻²), 18 to 20° C. day and 14 to 16° C.night temperature, 50 to 70% relative air humidity and 3) pest control:sulphur spray every 4 to 6 weeks and biological control of thrips usingAmblyseius caliginosus (Novartis BCM Ltd, UK).

[0234] Isolation of Explants and Culture Initiation

[0235] Two sources of primary explants were used; scutellar andinflorescence tissues. For scutella, early-medium milk stage grainscontaining immature translucent embryos were harvested andsurface-sterilized in 70% ethanol for 5 min and 0.5% hypochloritesolution for 15-30 min. For inflorescences, tillers containing 0.5-1.0cm inflorescences were harvested by cutting below theinflorescence-bearing node (the second node of a tiller). The tillerswere trimmed to approximately 8-10 cm length and surface-sterilized asabove with the upper end sealed with Nescofilm (Bando Chemical Ind. Ltd,Japan).

[0236] Under aseptic conditions, embryos of approximately 0.5-1.0 mmlength were isolated and the embryo axis removed. Inflorescences weredissected from the tillers and cut into approximately 1 mm pieces.Thirty scutella or 1 mm inflorescence explants were placed in the center(18 mm target circle) of a 90 mm Petri dish containing MD0.5 or L7D2culture medium. Embryos were placed with the embryo-axis side in contactwith the medium exposing the scutellum to bombardment whereasinflorescence pieces were placed randomly. Cultures were incubated at25±° C. in darkness for approximately 24 h before bombardment. Afterbombardment, explants from each bombarded plate were spread across threeplates for callus induction.

[0237] Culture Media

[0238] The standard callus induction medium for scutellar tissues(MD0.5) consisted of solidified (0.5% Agargel, Sigma A3301) modified MSmedium supplemented with 9% sucrose, 10 mg I⁻¹ AgNO₃ and 0.5 mg I⁻¹2,4-D (Rasco-Gaunt et al., 1999). Inflorescence tissues were cultured onL7D2 which consisted of solidified (0.5% Agargel) L3 medium supplementedwith 9% maltose and 2 mg I⁻¹ 2,4-D (Rasco-Gaunt and Barcelo, 1999). Thebasal shoot induction medium, RZ contained L salts, vitamins andinositol, 3% w/v maltose, 0.1 mg I⁻¹ 2,4-D and 5 mg I⁻¹ zeatin(Rasco-Gaunt and Barcelo, 1999). Regenerated plantlets were maintainedin RO medium with the same composition as RZ, but without 2,4-D andzeatin.

[0239] DNA Precipitation Procedure and Particle Bombardment

[0240] Submicron gold particles (0.6 μm Micron Gold, Bio-Rad) werecoated with a plasmid containing the maize In-2:LEC1 construct followingthe protocol modified from the original Bio-Rad procedure (Barcelo andLazzeri, 1995). The standard precipitation mixture consisted of 1 mg ofgold particles in 50 μl SDW, 50 μl of 2.5 M calcium chloride, 20 μl of100 mM spermidine free base and 5 μl DNA (concentration 1 μg μl⁻¹).After combining the components, the mixture was vortexed and thesupernatant discarded. The particles were then washed with 150 μlabsolute ethanol and finally resuspended in 85 μl absolute ethanol. TheDNA/gold ethanol solution was kept on ice to minimise ethanolevaporation. For each bombardment, 5 μl of DNA/gold ethanol solution(ca. 60 μg gold) was loaded onto the macrocarrier.

[0241] Particle bombardments were carried out using DuPont PDS 1000/Hegun with a target distance of 5.5 cm from the stopping plate at 650 psiacceleration pressure and 28 in. Hg chamber vacuum pressure.

[0242] Regeneration of Transformants

[0243] For callus induction, bombarded explants were distributed overthe surface of the medium in the original dish and two other dishes andcultured at 25±1° C. in darkness for three weeks. Development of somaticembryos from each callus were periodically recorded. For shootinduction, calluses were transferred to RZ medium and cultured under 12h light (250 μE s⁻¹ m⁻², from cool white fluorescent tubes) at 25±1° C.for three weeks for two rounds. All plants regenerating from the samecallus were noted. Plants growing more vigorously than the controlcultures were potted in soil after 6-9 weeks in R0 medium. The plantletswere acclimatized in a propagator for 1-2 weeks. Thereafter, the plantswere grown to maturity under growth conditions described above.

[0244] DNA Isolation from Callus and Leaf Tissues

[0245] Genomic DNA was extracted from calluses or leaves using amodification of the CTAB (cetyltriethylammonium bromide, Sigma H5882)method described by Stacey and Isaac (1994). Approximately 100-200 mg offrozen tissues was ground into powder in liquid nitrogen and homogenisedin 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA, 0.1 M Tris-ClpH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65° C. Homogenised sampleswere allowed to cool at room temperature for 15 min before a singleprotein extraction with approximately 1 ml 24:1 v/v chloroform:octanolwas done. Samples were centrifuged for 7 min at 13,000 rpm and the upperlayer of supernatant collected using wide-mouthed pipette tips. DNA wasprecipitated from the supernatant by incubation in 95% ethanol on icefor 1 h. DNA threads were spooled onto a glass hook, washed in 75%ethanol containing 0.2 M sodium acetate for 10 min, air-dried for 5 minand resuspended in TE buffer. Five μl RNAse A was added to the samplesand incubated at 37° C. for 1 h.

[0246] For quantification of genomic DNA, gel electrophoresis wasperformed using an 0.8% agarose gel in 1×TBE buffer. One microlitre ofthe samples were fractionated alongside 200, 400, 600 and 800 ng μl⁻¹ λuncut DNA markers.

[0247] Polymerase Chain Reaction (PCR) Analysis

[0248] The presence of the maize LEC1 polynucleotide was analyzed by PCRusing 100-200 ng template DNA in a 30 ml PCR reaction mixture containing1× concentration enzyme buffer (10 mM Tris-HCl pH 8.8, 1.5 mM magnesiumchloride, 50 mM potassium chloride, 0.1% Triton X-100), 200 μM dNTPs,0.3 μM primers and 0.022 U TaqDNA polymerase (Boehringer Mannheim).Thermocycling conditions were as follows (30 cycles): denaturation at95° C. for 30 s, annealing at 55° C. for 1 min and extension at 72° C.for 1 min. Primer sequences (F=forward; R=reverse) used were: (F) 5′-CGCTCT GTC ACC TGT TGT ACT C-3′ (R) 5′-CGT GAT GAA GCT GAT GTA CTC C-3′.Approximate PCR product length was 620 bp.

[0249] Results

[0250] Following experiments to show increased regeneration capacity andimprovement of maize transformation frequencies by expression of maizeLEC1, the polynucleotide was then introduced into wheat scutellar andinflorescence explants, driven by the maize In2 promoter. Both tissuesare used for wheat transformation.

[0251] Subsequent to the induction of somatic embryos from both tissuesafter three weeks on a 2,4-D-containing induction medium, calluses wereassessed prior to transfer onto shoot regeneration medium. Callusassessment involved: a) scoring calluses as 0=non-embryogenic callus,1=25%, 2=25-50%, 3=50-75%, 4=75-100% of callus surface embryogenic, andb) determining embryogenic capacity expressed in percentage as thenumber of embryogenic calluses/total number of calluses (scutella orinflorescence) assessed.

[0252] Scutellum Calluses

[0253] Mean callus scores of control (1.4±0.3) and LEC-bombarded(1.4±0.3) scutellar tissues of wheat line NH535 were not significantlydifferent. However, callus score of LEC-bombarded scutella (1.5±0.5) ofwheat line BO 014 was significantly improved in comparison with thecontrol (0.5±0.2). Similarly, embryogenic capacity of line NH535 did notseem to be affected by LEC treatment (LEC calluses=84.3±9.3%, controlcalluses=90%). However, LEC-bombarded line BO 014 had clear increases inthe embryogenic callus frequency (LEC calluses=75.4±16.8%, controlcalluses=36.7±4.7%). Examining the quality of embryogenic callusesformed, both lines showed significant increases in the number of ‘good’calluses produced i.e. calluses with scores of 3 or 4. ‘Good’ qualitycallus of line NH535 increased from 5 to 22.3% whilst line BO 014increased from 0 to 23.6%. These calluses were generally large, rapidlygrowing and vigorous.

[0254] After callus induction and assessment, calluses were transferredonto shoot induction media for a total of six weeks. Shoot regenerationof calluses was determined, as the number of shoot regeneratingcalluses/total number of calluses assessed (expressed as percentages).Shoot regeneration of cultures corresponded with the quality andquantity of somatic embryos produced in each callus. Hence, regenerationof LEC-bombarded (71.9±12.1) and control (70±14) callus tissues of lineNH535 were not significantly different. However, regeneration ofLEC-bombarded calluses (52.3±26.9) of wheat line BO 014 wassignificantly improved in comparison with the control (15.6±6.3).

[0255] To test the suitability of LEC as a positive selection system forwheat, sample tissues from vigorous calluses were analyzed for thepresence of LEC sequences. Forty-one BO 014 and 13 NH535 calluses wereselected. The results were that 10/41 BO 014 calluses and 8/13 NH535were PCR positive. Thus, transformed lines were identified withoutselection at frequencies of 24.4% and 61.5%. These frequencies arecomparable with conventional selection systems such as herbicide- andantibiotic-resistance systems (e.g. bar, nptII) applied in wheattransformation where selection ‘escape’ frequencies are commonly highand variable. Furthermore, we know of no other report of wheattransformation by morphological selection in the absence of a selectionagent.

[0256] Callus transformation frequencies were 5.6% and 4.4% in NH535 andBO 014 lines, respectively. Transgenic plants were also recovered fromLEC-positive callus lines. Seven non-clonal plants were recovered fromNH535 and six non-clonal plants were recovered from BO 014 to give planttransformation frequencies of 3.9 and 3.3%, respectively, based on thenumber of explants bombarded.

[0257] Inflorescence Calluses

[0258] The use of inflorescence tissues as explants for the tissueculture and transformation of wheat offer several advantages over seedexplants such as scutella (Rasco-Gaunt and Barcelo, 1999). However,responses of these tissues to culture are highly genotype-dependent andcalluses are often non-regenerative despite having a‘highly-embryogenic’ appearance. Hence, LEC was introduced intoinflorescence tissues to see whether regeneration could be enhanced on apoorly regenerating line such BO 014.

[0259] Using line BO 014, shoot regeneration was significantly improvedin LEC-bombarded tissues, although callus quality appeared similar inbombarded and control tissues. Whereas no shoot was regenerated fromcontrol cultures, eight plants were regenerated from LEC-bombardedcalluses to give a shoot regeneration frequency of 10.7%. Summary oftissue culture data Mean % Good callus Embryo. calluses Wheat scoreCapacity (score Regeneration line Treatment (0-4) (%) 3 & 4) (%) NH 535Control 1.4 ± 0.3 90.0 5   70 ± 14 LEC1 1.4 ± 0.3 84.3 ± 9.3 22.3 71.9 ±12.1 BO 014 Control 0.5 ± 0.2 36.7 ± 4.7 0 15.6 ± 6.3 LEC1 1.5 ± 0.575.4 ± 16.8 23.6 52.3 ± 26.9

[0260] Type of callus produced per treatment per line NH535 BO014 CallusScore Control LEC1 Control LEC1 0 10 15.7 63.2 25 1 45 43.3 26.3 30 2 4028.7 10.5 21.4 3 5 11.2 0 17.9 4 0 1.1 0 5.7

[0261] Transformation Frequency Wheat line Callus line Plant line(non-clonal) NH 535 10/180 (5.6%) 7/180 (3.9%) BO 014  8/180 (4.4%)6/180 (3.3%)

Example 15 Expression of Chimeric Genes in Dicot Cells

[0262] The LEC1 polynucleotide can also be used to improve thetransformation of soybean. To demonstrate this the construct consistingof the In2 promoter and LEC1 coding region were introduced intoembryogenic suspension cultures of soybean by particle bombardment usingessentially the methods described in Parrott, W. A., L. M. Hoffman, D.F. Hildebrand, E. G. Williams, and G. B. Collins, (1989) Recovery ofprimary transformants of soybean, Plant Cell Rep. 7:615-617. This methodwith modifications is described below.

[0263] Seed was removed from pods when the cotyledons were between 3 and5 mm in length. The seeds were sterilized in a Chlorox solution (0.5%)for 15 minutes after which time the seeds were rinsed with steriledistilled water. The immature cotyledons were excised by first cuttingaway the portion of the seed that contains the embryo axis. Thecotyledons were then removed from the seed coat by gently pushing thedistal end of the seed with the blunt end of the scalpel blade. Thecotyledons were then placed (flat side up) SB1 initiation medium (MSsalts, B5 vitamins, 20 mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH5.8). The Petri plates were incubated in the light (16 hr day; 75-80 μE)at 26° C. After 4 weeks of incubation the cotyledons were transferred tofresh SB1 medium. After an additional two weeks, globular stage somaticembryos that exhibit proliferative areas were excised and transferred toFN Lite liquid medium (Samoylov, V. M., D. M. Tucker, and W. A. Parrott(1998) Soybean [Glycine max (L.) Merrill] embryogenic cultures: the roleof sucrose and total nitrogen content on proliferation. In Vitro CellDev. Biol.- Plant 34:8-13). About 10 to 12 small clusters of somaticembryos were placed in 250 ml flasks containing 35 ml of SB172 medium.The soybean embryogenic suspension cultures were maintained in 35 mLliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights (20 μE) on a 16:8 hour day/night schedule. Cultures weresub-cultured every two weeks by inoculating approximately 35 mg oftissue into 35 mL of liquid medium.

[0264] Soybean embryogenic suspension cultures were then be transformedusing particle gun bombardment (Klein et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A BioRad Biolistic™ PDS1000/HEinstrument was used for these transformations. A selectable marker genewhich was used to facilitate soybean transformation is a chimeric genecomposed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al.(1985) Nature 313:810-812), the hygromycin phosphotransferase gene fromplasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188) andthe 3′ region of the nopaline synthase gene from the T-DNA of the Tiplasmid of Agrobacterium tumefaciens.

[0265] To 50 μL of a 60 mg/mL 1 μm gold particle suspension was added(in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μLCaCl₂ (2.5 M). The particle preparation was agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles were washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensionwas sonicated three times for one second each. Five μL of the DNA-coatedgold particles were then loaded on each macro carrier disk.

[0266] Approximately 300-400 mg of a two-week-old suspension culture wasplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. Membrane rupture pressure was set at1100 psi and the chamber was evacuated to a vacuum of 28 inches mercury.The tissue was placed approximately 8 cm away from the retaining screen,and was bombarded three times. Following bombardment, the tissue wasdivided in half and placed back into 35 ml of FN Lite medium.

[0267] Five to seven days after bombardment, the liquid medium wasexchanged with fresh medium. Eleven days post bombardment the medium wasexchanged with fresh medium containing 50 mg/mL hygromycin. Thisselective medium was refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue was observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue wasremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each new linewas treated as an independent transformation event. These suspensionswere then subcultured and maintained as clusters of immature embryos, ortissue was regenerated into whole plants by maturation and germinationof individual embryos.

[0268] Two different genotypes were used in these experiments: 92B91 and93B82. Samples of tissue were either bombarded with the hygromycinresistance gene alone or with a 1:1 mixture of the hygromycin resistancegene and the LEC1 construct. Embryogenic cultures generated from 92B91generally produce transformation events while cultures from 93B82 aremuch more difficult to transform. For transformation experiments with92B91, approximately equal numbers of transformants were recovered frombombardments conducted with the LEC1 polynucleotide as without it.Twenty-nine transformants were recovered from the LEC1-treated 92B91tissue while 27 transformants were recovered from tissue receiving onlythe hygromycin resistance gene. In contrast, transformants were onlyrecovered from 93B82 tissue receiving the LEC1 polynucleotide (none wererecovered from the treatment using only the hygromycin resistance gene).Five transformants were recovered from 93B82 tissue bombarded with theLEC1 polynucleotide while no transformants were recovered from tissuetreated with only the hygromycin resistance gene. These results showthat the LEC1 polynucleotide will be very valuable for gene transfer torecalcitrant genotypes of soybean.

1 26 1 1173 DNA Zea mays CDS (69)...(902) 1 ccacgcgtcc gccaccacaccacgagcgcg cgataaccct agctagcttc aggtagtagc 60 gagagcca atg gac tcc agcagc ttc ctc cct gcc gcc ggc gcg gag aat 110 Met Asp Ser Ser Ser Phe LeuPro Ala Ala Gly Ala Glu Asn 1 5 10 ggc tcg gcg gcg ggc ggc gcc aac aatggc ggc gct gct cag cag cat 158 Gly Ser Ala Ala Gly Gly Ala Asn Asn GlyGly Ala Ala Gln Gln His 15 20 25 30 gcg gcg ccg gcg atc cgc gag cag gaccgg ctg atg ccg atc gcg aac 206 Ala Ala Pro Ala Ile Arg Glu Gln Asp ArgLeu Met Pro Ile Ala Asn 35 40 45 gtg atc cgc atc atg cgg cgc gtg ctg ccggcg cac gcc aag atc tcg 254 Val Ile Arg Ile Met Arg Arg Val Leu Pro AlaHis Ala Lys Ile Ser 50 55 60 gac gac gcc aag gag acg atc cag gag tgc gtgtcg gag tac atc agc 302 Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys Val SerGlu Tyr Ile Ser 65 70 75 ttc atc acg ggg gag gcc aac gag cgg tgc cag cgggag cag cgc aag 350 Phe Ile Thr Gly Glu Ala Asn Glu Arg Cys Gln Arg GluGln Arg Lys 80 85 90 acc atc acc gcc gag gac gtg ctg tgg gcc atg agc cgcctc ggc ttc 398 Thr Ile Thr Ala Glu Asp Val Leu Trp Ala Met Ser Arg LeuGly Phe 95 100 105 110 gac gac tac gtc gag ccg ctc ggc gcc tac ctc caccgc tac cgc gag 446 Asp Asp Tyr Val Glu Pro Leu Gly Ala Tyr Leu His ArgTyr Arg Glu 115 120 125 ttc gag ggc gac gcg cgc ggc gtc ggg ctc gtc ccgggg gcc gcc cca 494 Phe Glu Gly Asp Ala Arg Gly Val Gly Leu Val Pro GlyAla Ala Pro 130 135 140 tcg cgc ggc ggc gac cac cac ccg cac tcc atg tcgcca gcg gcg atg 542 Ser Arg Gly Gly Asp His His Pro His Ser Met Ser ProAla Ala Met 145 150 155 ctc aag tcc cgc ggg cca gtc tcc gga gcc gcc atgcta ccg cac cac 590 Leu Lys Ser Arg Gly Pro Val Ser Gly Ala Ala Met LeuPro His His 160 165 170 cac cac cac cac gac atg cag atg cac gcc gcc atgtac ggg gga acg 638 His His His His Asp Met Gln Met His Ala Ala Met TyrGly Gly Thr 175 180 185 190 gcc gtg ccc ccg ccg gcc ggg cct cct cac cacggc ggg ttc ctc atg 686 Ala Val Pro Pro Pro Ala Gly Pro Pro His His GlyGly Phe Leu Met 195 200 205 cca cac cca cag ggt agt agc cac tac ctg ccttac gcg tac gag ccc 734 Pro His Pro Gln Gly Ser Ser His Tyr Leu Pro TyrAla Tyr Glu Pro 210 215 220 acg tac ggc ggt gag cac gcc atg gct gca tactat gga ggc gcc gcg 782 Thr Tyr Gly Gly Glu His Ala Met Ala Ala Tyr TyrGly Gly Ala Ala 225 230 235 tac gcg ccc ggc aac ggc ggg agc ggc gac ggcagt ggc agt ggc ggc 830 Tyr Ala Pro Gly Asn Gly Gly Ser Gly Asp Gly SerGly Ser Gly Gly 240 245 250 ggt ggc ggg agc gcg tcg cac aca ccg cag ggcagc ggc ggc ttg gag 878 Gly Gly Gly Ser Ala Ser His Thr Pro Gln Gly SerGly Gly Leu Glu 255 260 265 270 cac ccg cac ccg ttc gcg tac aagtagctagttc gtacgtcgtt cgacttgagc 932 His Pro His Pro Phe Ala Tyr Lys 275aagccatcga tctgctgatc tgaacgtacg ctgtattgta cacgcatgca cgtacgtatc 992ggcggctagc tctcctgttt aagttgtact gtgattctgt cccggccggc tagcaactta 1052gtatcttcct tcagtctcta gtttcttagc agtcgtagaa gtgttcaatg cttgccagtg 1112tgttgtttta gggccggggt aaaccatccg atgagattat ttcaaaaaaa aaaaaaaaaa 1172 a1173 2 278 PRT Zea mays 2 Met Asp Ser Ser Ser Phe Leu Pro Ala Ala GlyAla Glu Asn Gly Ser 1 5 10 15 Ala Ala Gly Gly Ala Asn Asn Gly Gly AlaAla Gln Gln His Ala Ala 20 25 30 Pro Ala Ile Arg Glu Gln Asp Arg Leu MetPro Ile Ala Asn Val Ile 35 40 45 Arg Ile Met Arg Arg Val Leu Pro Ala HisAla Lys Ile Ser Asp Asp 50 55 60 Ala Lys Glu Thr Ile Gln Glu Cys Val SerGlu Tyr Ile Ser Phe Ile 65 70 75 80 Thr Gly Glu Ala Asn Glu Arg Cys GlnArg Glu Gln Arg Lys Thr Ile 85 90 95 Thr Ala Glu Asp Val Leu Trp Ala MetSer Arg Leu Gly Phe Asp Asp 100 105 110 Tyr Val Glu Pro Leu Gly Ala TyrLeu His Arg Tyr Arg Glu Phe Glu 115 120 125 Gly Asp Ala Arg Gly Val GlyLeu Val Pro Gly Ala Ala Pro Ser Arg 130 135 140 Gly Gly Asp His His ProHis Ser Met Ser Pro Ala Ala Met Leu Lys 145 150 155 160 Ser Arg Gly ProVal Ser Gly Ala Ala Met Leu Pro His His His His 165 170 175 His His AspMet Gln Met His Ala Ala Met Tyr Gly Gly Thr Ala Val 180 185 190 Pro ProPro Ala Gly Pro Pro His His Gly Gly Phe Leu Met Pro His 195 200 205 ProGln Gly Ser Ser His Tyr Leu Pro Tyr Ala Tyr Glu Pro Thr Tyr 210 215 220Gly Gly Glu His Ala Met Ala Ala Tyr Tyr Gly Gly Ala Ala Tyr Ala 225 230235 240 Pro Gly Asn Gly Gly Ser Gly Asp Gly Ser Gly Ser Gly Gly Gly Gly245 250 255 Gly Ser Ala Ser His Thr Pro Gln Gly Ser Gly Gly Leu Glu HisPro 260 265 270 His Pro Phe Ala Tyr Lys 275 3 20 DNA Artificial Sequenceprimer 3 tagtagcgag agccaatgga 20 4 20 DNA Artificial Sequence primer 4gccgggacag aatcacagta 20 5 20 DNA Artificial Sequence primer 5tagtagcgag agccaatgga 20 6 20 DNA Artificial Sequence primer 6cccggcccta aaacaacaca 20 7 481 DNA Argemone mexicana CDS (44)...(481)misc_feature (1)...(481) n = A,T,C or G 7 cgagagaaag agttggtgaagaagaagaag aagttgaaaa gag atg gaa cgt ggt 55 Met Glu Arg Gly 1 ggt ggtggt ggt ggt agt ggt ggt ggt ttc cat gga tat cag aaa ctc 103 Gly Gly GlyGly Gly Ser Gly Gly Gly Phe His Gly Tyr Gln Lys Leu 5 10 15 20 cca aaatca aac tcc gct gga atg atg ctc tcg gag cta tcg aat aac 151 Pro Lys SerAsn Ser Ala Gly Met Met Leu Ser Glu Leu Ser Asn Asn 25 30 35 aac aac aatatt gac gta aac tct aca tgt act gta cga gag caa gat 199 Asn Asn Asn IleAsp Val Asn Ser Thr Cys Thr Val Arg Glu Gln Asp 40 45 50 cga tac atg ccaatt gct aat gtg atc agg atc atg cgt aag gta ctt 247 Arg Tyr Met Pro IleAla Asn Val Ile Arg Ile Met Arg Lys Val Leu 55 60 65 cct act cat gcc aagatc tct gac gat gcc aaa gaa act atc caa gaa 295 Pro Thr His Ala Lys IleSer Asp Asp Ala Lys Glu Thr Ile Gln Glu 70 75 80 tgt gtc tca gaa tac atcagt ttc atc aca agt gaa gcc aat gat cgt 343 Cys Val Ser Glu Tyr Ile SerPhe Ile Thr Ser Glu Ala Asn Asp Arg 85 90 95 100 tgc caa cgt gaa caa agaaag aca atc aca gct gaa gat gtt tta tgg 391 Cys Gln Arg Glu Gln Arg LysThr Ile Thr Ala Glu Asp Val Leu Trp 105 110 115 gcg atg agc aaa cta gggntt gat gag tac att gaa cct cta act ctt 439 Ala Met Ser Lys Leu Gly XaaAsp Glu Tyr Ile Glu Pro Leu Thr Leu 120 125 130 tac ctt caa cgt tat cgtgag ttt gaa ggt gna cgt tgg tca 481 Tyr Leu Gln Arg Tyr Arg Glu Phe GluGly Xaa Arg Trp Ser 135 140 145 8 146 PRT Argemone mexicana VARIANT(1)...(146) Xaa = Any Amino Acid 8 Met Glu Arg Gly Gly Gly Gly Gly GlySer Gly Gly Gly Phe His Gly 1 5 10 15 Tyr Gln Lys Leu Pro Lys Ser AsnSer Ala Gly Met Met Leu Ser Glu 20 25 30 Leu Ser Asn Asn Asn Asn Asn IleAsp Val Asn Ser Thr Cys Thr Val 35 40 45 Arg Glu Gln Asp Arg Tyr Met ProIle Ala Asn Val Ile Arg Ile Met 50 55 60 Arg Lys Val Leu Pro Thr His AlaLys Ile Ser Asp Asp Ala Lys Glu 65 70 75 80 Thr Ile Gln Glu Cys Val SerGlu Tyr Ile Ser Phe Ile Thr Ser Glu 85 90 95 Ala Asn Asp Arg Cys Gln ArgGlu Gln Arg Lys Thr Ile Thr Ala Glu 100 105 110 Asp Val Leu Trp Ala MetSer Lys Leu Gly Xaa Asp Glu Tyr Ile Glu 115 120 125 Pro Leu Thr Leu TyrLeu Gln Arg Tyr Arg Glu Phe Glu Gly Xaa Arg 130 135 140 Trp Ser 145 9942 DNA Glycine max CDS (3)...(722) 9 gc acg agc tct ctt ata atc aca cacaca cct acc tta ata gct atg 47 Thr Ser Ser Leu Ile Ile Thr His Thr ProThr Leu Ile Ala Met 1 5 10 15 gaa act gga ggc ttt cac ggc tac cgc aagctc ccc aac acc acc gct 95 Glu Thr Gly Gly Phe His Gly Tyr Arg Lys LeuPro Asn Thr Thr Ala 20 25 30 ggg ttg aag ctg tca gtg tca gac atg aac atgagg cag cag gta gca 143 Gly Leu Lys Leu Ser Val Ser Asp Met Asn Met ArgGln Gln Val Ala 35 40 45 tca tca gat cac agt gca gcc aca gga gag gag aacgaa tgc acg gtg 191 Ser Ser Asp His Ser Ala Ala Thr Gly Glu Glu Asn GluCys Thr Val 50 55 60 agg gag caa gac agg ttc atg cca atc gcc aac gtg attagg atc atg 239 Arg Glu Gln Asp Arg Phe Met Pro Ile Ala Asn Val Ile ArgIle Met 65 70 75 cgc aag att ctc cct cca cac gca aaa atc tcg gac gat gcaaaa gaa 287 Arg Lys Ile Leu Pro Pro His Ala Lys Ile Ser Asp Asp Ala LysGlu 80 85 90 95 aca atc caa gag tgc gtg tct gag tac atc agc ttc atc acaggt gag 335 Thr Ile Gln Glu Cys Val Ser Glu Tyr Ile Ser Phe Ile Thr GlyGlu 100 105 110 gcg aac gag cgt tgc cag agg gag cag cgg aag acc ata accgca gag 383 Ala Asn Glu Arg Cys Gln Arg Glu Gln Arg Lys Thr Ile Thr AlaGlu 115 120 125 gac gtg ctt tgg gcc atg agc aag ctt gga ttc gac gac tacatc gaa 431 Asp Val Leu Trp Ala Met Ser Lys Leu Gly Phe Asp Asp Tyr IleGlu 130 135 140 ccg ttg acc atg tac ctt cac cgc tac cgt gaa ctt gag ggtgac cgc 479 Pro Leu Thr Met Tyr Leu His Arg Tyr Arg Glu Leu Glu Gly AspArg 145 150 155 acc tct atg agg ggt gaa cca ctc ggg aag agg act gtg gaatac gcc 527 Thr Ser Met Arg Gly Glu Pro Leu Gly Lys Arg Thr Val Glu TyrAla 160 165 170 175 acg ctt ggt gtt gct act gct ttt gtc cct cca ccc tatcat cac cac 575 Thr Leu Gly Val Ala Thr Ala Phe Val Pro Pro Pro Tyr HisHis His 180 185 190 aat ggg tac ttt ggt gct gcc atg ccc atg ggg act tacgtt agg gaa 623 Asn Gly Tyr Phe Gly Ala Ala Met Pro Met Gly Thr Tyr ValArg Glu 195 200 205 gcg cca cca aat aca gcc tcc tcc cat cac cac cac caccac cac cac 671 Ala Pro Pro Asn Thr Ala Ser Ser His His His His His HisHis His 210 215 220 cac cat gct cgt gga atc tcc aat gct cat gaa cca aatgct cgc tcc 719 His His Ala Arg Gly Ile Ser Asn Ala His Glu Pro Asn AlaArg Ser 225 230 235 ata taaaattata taattatgac taggattcag aacaagacttgatgatgatt 772 Ile 240 agcttaactc tcagtaattg gtgctagagt actactgttgttgaggatac tttattttat 832 aattaagggc tgggaaggga gttagtatat tcctaatcctaactatgtgc atctttaatt 892 tatgaaatca ctttgtttta acctttgatg aaaaaaaaaaaaaaaaaaaa 942 10 240 PRT Glycine max 10 Thr Ser Ser Leu Ile Ile Thr HisThr Pro Thr Leu Ile Ala Met Glu 1 5 10 15 Thr Gly Gly Phe His Gly TyrArg Lys Leu Pro Asn Thr Thr Ala Gly 20 25 30 Leu Lys Leu Ser Val Ser AspMet Asn Met Arg Gln Gln Val Ala Ser 35 40 45 Ser Asp His Ser Ala Ala ThrGly Glu Glu Asn Glu Cys Thr Val Arg 50 55 60 Glu Gln Asp Arg Phe Met ProIle Ala Asn Val Ile Arg Ile Met Arg 65 70 75 80 Lys Ile Leu Pro Pro HisAla Lys Ile Ser Asp Asp Ala Lys Glu Thr 85 90 95 Ile Gln Glu Cys Val SerGlu Tyr Ile Ser Phe Ile Thr Gly Glu Ala 100 105 110 Asn Glu Arg Cys GlnArg Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp 115 120 125 Val Leu Trp AlaMet Ser Lys Leu Gly Phe Asp Asp Tyr Ile Glu Pro 130 135 140 Leu Thr MetTyr Leu His Arg Tyr Arg Glu Leu Glu Gly Asp Arg Thr 145 150 155 160 SerMet Arg Gly Glu Pro Leu Gly Lys Arg Thr Val Glu Tyr Ala Thr 165 170 175Leu Gly Val Ala Thr Ala Phe Val Pro Pro Pro Tyr His His His Asn 180 185190 Gly Tyr Phe Gly Ala Ala Met Pro Met Gly Thr Tyr Val Arg Glu Ala 195200 205 Pro Pro Asn Thr Ala Ser Ser His His His His His His His His His210 215 220 His Ala Arg Gly Ile Ser Asn Ala His Glu Pro Asn Ala Arg SerIle 225 230 235 240 11 905 DNA Veronia mespilifolia CDS (58)...(699) 11gcacgagcca atttctagag agagaacgag agagaattct ctaaagagga aaaatag atg 60Met 1 gaa cgt gga gga ggt ttc cat ggc tac cac agg ctc ccc atc cac cct108 Glu Arg Gly Gly Gly Phe His Gly Tyr His Arg Leu Pro Ile His Pro 5 1015 aca tct gga atc caa caa tcg gat atg aag cta aag cta cca gaa atg 156Thr Ser Gly Ile Gln Gln Ser Asp Met Lys Leu Lys Leu Pro Glu Met 20 25 30acc aac aat aac tcg tcc act gat gac aat gag tgc acc gtt cga gaa 204 ThrAsn Asn Asn Ser Ser Thr Asp Asp Asn Glu Cys Thr Val Arg Glu 35 40 45 caggac cgc ttc atg ccg ata gca aac gtg atc cgc atc atg cgg aag 252 Gln AspArg Phe Met Pro Ile Ala Asn Val Ile Arg Ile Met Arg Lys 50 55 60 65 atcctt cct cca cat gcc aag atc tct gat gat gcc aaa gag acg atc 300 Ile LeuPro Pro His Ala Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile 70 75 80 caa gaatgt gtt tca gag tac att agc ttt gtc aca ggc gag gca aat 348 Gln Glu CysVal Ser Glu Tyr Ile Ser Phe Val Thr Gly Glu Ala Asn 85 90 95 gac cgc tgccag cgt gag caa agg aag acc atc aca gct gaa gat gtg 396 Asp Arg Cys GlnArg Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp Val 100 105 110 ctc tgg gctatg agc aaa ctg gga ttt gat gat tat atc gag ccc ttg 444 Leu Trp Ala MetSer Lys Leu Gly Phe Asp Asp Tyr Ile Glu Pro Leu 115 120 125 act gtg tatctc cat cgc tac agg gag ttt gat ggt ggc gaa cgt gga 492 Thr Val Tyr LeuHis Arg Tyr Arg Glu Phe Asp Gly Gly Glu Arg Gly 130 135 140 145 tcc ataagg ggt gag ccc ctt gtg aag agg agt act tct gat cct ggt 540 Ser Ile ArgGly Glu Pro Leu Val Lys Arg Ser Thr Ser Asp Pro Gly 150 155 160 cac tttggg atg gct tct ttt gtg cct gct ttt cat atg ggt cat cat 588 His Phe GlyMet Ala Ser Phe Val Pro Ala Phe His Met Gly His His 165 170 175 aac ggcttc ttt ggt cct gca agc att ggt ggt ttc ctg aaa gac cca 636 Asn Gly PhePhe Gly Pro Ala Ser Ile Gly Gly Phe Leu Lys Asp Pro 180 185 190 tcg agtgct ggc cct tcg gga cct gca gtc gct ggg ttt gag ccg tat 684 Ser Ser AlaGly Pro Ser Gly Pro Ala Val Ala Gly Phe Glu Pro Tyr 195 200 205 gct cagtgt aaa gag taactgcaaa aagtaggggt tgggatgaga tgatgatgat 739 Ala Gln CysLys Glu 210 ggtggtggtg gtggtggttt gttttgtttt gttctttctt ttttttttcttctttctttt 799 cttggtcatt gaggaacaaa cttacattgg ttcactttgg ctaggcatgtaaacggttaa 859 catgcttatc aagtagtagt tttcgatcaa aaaaaaaaaa aaaaaa 905 12214 PRT Veronia mespilifolia 12 Met Glu Arg Gly Gly Gly Phe His Gly TyrHis Arg Leu Pro Ile His 1 5 10 15 Pro Thr Ser Gly Ile Gln Gln Ser AspMet Lys Leu Lys Leu Pro Glu 20 25 30 Met Thr Asn Asn Asn Ser Ser Thr AspAsp Asn Glu Cys Thr Val Arg 35 40 45 Glu Gln Asp Arg Phe Met Pro Ile AlaAsn Val Ile Arg Ile Met Arg 50 55 60 Lys Ile Leu Pro Pro His Ala Lys IleSer Asp Asp Ala Lys Glu Thr 65 70 75 80 Ile Gln Glu Cys Val Ser Glu TyrIle Ser Phe Val Thr Gly Glu Ala 85 90 95 Asn Asp Arg Cys Gln Arg Glu GlnArg Lys Thr Ile Thr Ala Glu Asp 100 105 110 Val Leu Trp Ala Met Ser LysLeu Gly Phe Asp Asp Tyr Ile Glu Pro 115 120 125 Leu Thr Val Tyr Leu HisArg Tyr Arg Glu Phe Asp Gly Gly Glu Arg 130 135 140 Gly Ser Ile Arg GlyGlu Pro Leu Val Lys Arg Ser Thr Ser Asp Pro 145 150 155 160 Gly His PheGly Met Ala Ser Phe Val Pro Ala Phe His Met Gly His 165 170 175 His AsnGly Phe Phe Gly Pro Ala Ser Ile Gly Gly Phe Leu Lys Asp 180 185 190 ProSer Ser Ala Gly Pro Ser Gly Pro Ala Val Ala Gly Phe Glu Pro 195 200 205Tyr Ala Gln Cys Lys Glu 210 13 763 DNA Zea mays CDS (1)...(480) 13 gcacga ggc aag acc gtc acc tcc gag gac atc gtg tgg gcc atg agc 48 Ala ArgGly Lys Thr Val Thr Ser Glu Asp Ile Val Trp Ala Met Ser 1 5 10 15 cgcctc ggc ttc gac gac tac gtc gcg ccc ctc ggc gcc ttc ctc cag 96 Arg LeuGly Phe Asp Asp Tyr Val Ala Pro Leu Gly Ala Phe Leu Gln 20 25 30 cgc atgcgc gac gac agc gac cac ggc ggt gaa gag cgc ggc ggc cct 144 Arg Met ArgAsp Asp Ser Asp His Gly Gly Glu Glu Arg Gly Gly Pro 35 40 45 gca ggg cgtggt ggc tcg cgc cgc ggc tcg tcg tcc ttg ccg ctc cac 192 Ala Gly Arg GlyGly Ser Arg Arg Gly Ser Ser Ser Leu Pro Leu His 50 55 60 tgc ccg cag cagatg cac cac ctg cac cca gcc gtc tgc cgg cgt ccg 240 Cys Pro Gln Gln MetHis His Leu His Pro Ala Val Cys Arg Arg Pro 65 70 75 80 cac cag agc gtgtcg cct gct gca gga tac gcc gtc cgg ccc gtt ccc 288 His Gln Ser Val SerPro Ala Ala Gly Tyr Ala Val Arg Pro Val Pro 85 90 95 cgc ccg atg cca gcccgt ggg tac cgc atg cag ggc gga gac cac cgc 336 Arg Pro Met Pro Ala ArgGly Tyr Arg Met Gln Gly Gly Asp His Arg 100 105 110 agc gtg ggc ggc gtggct ccc tgc agc tac gga ggg gcg ctc gtc cag 384 Ser Val Gly Gly Val AlaPro Cys Ser Tyr Gly Gly Ala Leu Val Gln 115 120 125 gcc ggt gga acc caacac gtt gtt gga ttc cac gac gac gag gca agc 432 Ala Gly Gly Thr Gln HisVal Val Gly Phe His Asp Asp Glu Ala Ser 130 135 140 tct tcg agt gaa aatccg ccg ccg gag ggg cgt gcc gct ggc tcg aac 480 Ser Ser Ser Glu Asn ProPro Pro Glu Gly Arg Ala Ala Gly Ser Asn 145 150 155 160 tagcctagcttctcagttcc ccgtgtacaa taagaggggc ggtcgcggcg ccgcgccgcg 540 cccttgggttgggccgggcg ctatgctgca gtttggtttg taaactaacg agcctagggt 600 agctggtgcacgcgcgccac ctcgccggac gtcgccgtcg tcgtcggcat ggacttaacc 660 ggcgggccctgttgttattt ctcaagtttg tagccaacgc actgttcggt gcgttccata 720 atttaatttaccatgttgct ctcgaaaaaa aaaaaaaaaa aaa 763 14 160 PRT Zea mays 14 Ala ArgGly Lys Thr Val Thr Ser Glu Asp Ile Val Trp Ala Met Ser 1 5 10 15 ArgLeu Gly Phe Asp Asp Tyr Val Ala Pro Leu Gly Ala Phe Leu Gln 20 25 30 ArgMet Arg Asp Asp Ser Asp His Gly Gly Glu Glu Arg Gly Gly Pro 35 40 45 AlaGly Arg Gly Gly Ser Arg Arg Gly Ser Ser Ser Leu Pro Leu His 50 55 60 CysPro Gln Gln Met His His Leu His Pro Ala Val Cys Arg Arg Pro 65 70 75 80His Gln Ser Val Ser Pro Ala Ala Gly Tyr Ala Val Arg Pro Val Pro 85 90 95Arg Pro Met Pro Ala Arg Gly Tyr Arg Met Gln Gly Gly Asp His Arg 100 105110 Ser Val Gly Gly Val Ala Pro Cys Ser Tyr Gly Gly Ala Leu Val Gln 115120 125 Ala Gly Gly Thr Gln His Val Val Gly Phe His Asp Asp Glu Ala Ser130 135 140 Ser Ser Ser Glu Asn Pro Pro Pro Glu Gly Arg Ala Ala Gly SerAsn 145 150 155 160 15 622 DNA Zea mays CDS (3)...(622) misc_feature(1)...(622) n = A,T,C or G 15 gc atg aat aat ccc caa aac cct aaa gcc agtgct cct tgc acc ttg 47 Met Asn Asn Pro Gln Asn Pro Lys Ala Ser Ala ProCys Thr Leu 1 5 10 15 cca ccg gag ctt ccc aaa gaa gca gtg gcg acc gacgaa gca ccg ccg 95 Pro Pro Glu Leu Pro Lys Glu Ala Val Ala Thr Asp GluAla Pro Pro 20 25 30 cca atg ggc aac aac aac aac acg gaa tcg gcg acg gcgacg atg gtc 143 Pro Met Gly Asn Asn Asn Asn Thr Glu Ser Ala Thr Ala ThrMet Val 35 40 45 cgg gag cag gac cgg ctg atg ccc gtg gcc aac gtg tcc cgcatc atg 191 Arg Glu Gln Asp Arg Leu Met Pro Val Ala Asn Val Ser Arg IleMet 50 55 60 cgc caa gtg ctg cct ccg tac gcc aag atc tcc gac gac gcc cangaa 239 Arg Gln Val Leu Pro Pro Tyr Ala Lys Ile Ser Asp Asp Ala Xaa Glu65 70 75 gtn atc caa gaa ttg ctn ttc gga att tca tca ctt ncg tcc tgg cga287 Xaa Ile Gln Glu Leu Xaa Phe Gly Ile Ser Ser Leu Xaa Ser Trp Arg 8085 90 95 ggc gaa acg aag cgg tgc cac acc gag cgc cgc aag acc gtc acc tcc335 Gly Glu Thr Lys Arg Cys His Thr Glu Arg Arg Lys Thr Val Thr Ser 100105 110 gaa gac atc gtg tgg gcc atg agc cgc ctc ggc ttc gac gac tac gtc383 Glu Asp Ile Val Trp Ala Met Ser Arg Leu Gly Phe Asp Asp Tyr Val 115120 125 gcg ccc ctc ggc gcc ttc ctc cag cgc atg cgc gac nac agc gaa cac431 Ala Pro Leu Gly Ala Phe Leu Gln Arg Met Arg Asp Xaa Ser Glu His 130135 140 ggg ggt gaa aac gcg gcg gcc tgc ang ggg tng tgg tcn cgc cgc ggg479 Gly Gly Glu Asn Ala Ala Ala Cys Xaa Gly Xaa Trp Xaa Arg Arg Gly 145150 155 tcg tct nct tgg cgc tcc ctt gcc gca ana gat gac aac ttg cac caa527 Ser Ser Xaa Trp Arg Ser Leu Ala Ala Xaa Asp Asp Asn Leu His Gln 160165 170 175 acg tct gcc ggg ntc gga cca aaa ctn ttc cct gtt gca gga ataccc 575 Thr Ser Ala Gly Xaa Gly Pro Lys Xaa Phe Pro Val Ala Gly Ile Pro180 185 190 gtc cng ggc cnt tcc ccc ccn aat cca acc att tgg ttt ccc cttgc 622 Val Xaa Gly Xaa Ser Pro Xaa Asn Pro Thr Ile Trp Phe Pro Leu 195200 205 16 206 PRT Zea mays VARIANT (1)...(206) Xaa = Any Amino Acid 16Met Asn Asn Pro Gln Asn Pro Lys Ala Ser Ala Pro Cys Thr Leu Pro 1 5 1015 Pro Glu Leu Pro Lys Glu Ala Val Ala Thr Asp Glu Ala Pro Pro Pro 20 2530 Met Gly Asn Asn Asn Asn Thr Glu Ser Ala Thr Ala Thr Met Val Arg 35 4045 Glu Gln Asp Arg Leu Met Pro Val Ala Asn Val Ser Arg Ile Met Arg 50 5560 Gln Val Leu Pro Pro Tyr Ala Lys Ile Ser Asp Asp Ala Xaa Glu Xaa 65 7075 80 Ile Gln Glu Leu Xaa Phe Gly Ile Ser Ser Leu Xaa Ser Trp Arg Gly 8590 95 Glu Thr Lys Arg Cys His Thr Glu Arg Arg Lys Thr Val Thr Ser Glu100 105 110 Asp Ile Val Trp Ala Met Ser Arg Leu Gly Phe Asp Asp Tyr ValAla 115 120 125 Pro Leu Gly Ala Phe Leu Gln Arg Met Arg Asp Xaa Ser GluHis Gly 130 135 140 Gly Glu Asn Ala Ala Ala Cys Xaa Gly Xaa Trp Xaa ArgArg Gly Ser 145 150 155 160 Ser Xaa Trp Arg Ser Leu Ala Ala Xaa Asp AspAsn Leu His Gln Thr 165 170 175 Ser Ala Gly Xaa Gly Pro Lys Xaa Phe ProVal Ala Gly Ile Pro Val 180 185 190 Xaa Gly Xaa Ser Pro Xaa Asn Pro ThrIle Trp Phe Pro Leu 195 200 205 17 1121 DNA Glycine max CDS (3)...(1121)17 gc acg agg gaa act gga ggc ttt cat ggc tac cgc aag ctc ccc aac 47 ThrArg Glu Thr Gly Gly Phe His Gly Tyr Arg Lys Leu Pro Asn 1 5 10 15 acaacc tct ggg ttg aag ctg tca gtg tca gac atg aac atg aac atg 95 Thr ThrSer Gly Leu Lys Leu Ser Val Ser Asp Met Asn Met Asn Met 20 25 30 agg cagcag cag gta gca tca tca gat cag aac tgc agc aac cac agt 143 Arg Gln GlnGln Val Ala Ser Ser Asp Gln Asn Cys Ser Asn His Ser 35 40 45 gca gca ggagag gag aac gaa tgc acg gtg agg gag caa gac agg ttc 191 Ala Ala Gly GluGlu Asn Glu Cys Thr Val Arg Glu Gln Asp Arg Phe 50 55 60 atg cca atc gctaac gtg ata cgg atc atg cgc aag att ctc cct cca 239 Met Pro Ile Ala AsnVal Ile Arg Ile Met Arg Lys Ile Leu Pro Pro 65 70 75 cac gca aaa atc tccgat gat gca aag gag aca atc caa gag tgc gtg 287 His Ala Lys Ile Ser AspAsp Ala Lys Glu Thr Ile Gln Glu Cys Val 80 85 90 95 tcg gag tac atc agcttc atc acc ggg gag gcc aac gag cgt tgc cag 335 Ser Glu Tyr Ile Ser PheIle Thr Gly Glu Ala Asn Glu Arg Cys Gln 100 105 110 agg gag cag cgc aagacc ata acc gca gag gac gtg ctt tgg gca atg 383 Arg Glu Gln Arg Lys ThrIle Thr Ala Glu Asp Val Leu Trp Ala Met 115 120 125 agt aag ctt gga ttcgac gac tac atc gaa ccg tta acc atg tac ctt 431 Ser Lys Leu Gly Phe AspAsp Tyr Ile Glu Pro Leu Thr Met Tyr Leu 130 135 140 cac cgc tac cgt gagctg gag ggt gac cgc acc tct atg agg ggt gaa 479 His Arg Tyr Arg Glu LeuGlu Gly Asp Arg Thr Ser Met Arg Gly Glu 145 150 155 ccg ctc ggg aag aggact gtg gaa tat gcc acg ctt gct act gct ttt 527 Pro Leu Gly Lys Arg ThrVal Glu Tyr Ala Thr Leu Ala Thr Ala Phe 160 165 170 175 gtg ccg cca cccttt cat cac cac aat ggc tac ttt ggt gct gcc atg 575 Val Pro Pro Pro PheHis His His Asn Gly Tyr Phe Gly Ala Ala Met 180 185 190 ccc atg ggg acttac gtt agg gaa acg cca cca aat gct gcg tca tct 623 Pro Met Gly Thr TyrVal Arg Glu Thr Pro Pro Asn Ala Ala Ser Ser 195 200 205 cat cac cat catgga atc tcc aat gct cat gaa cca aat gct cgc tcc 671 His His His His GlyIle Ser Asn Ala His Glu Pro Asn Ala Arg Ser 210 215 220 ata taa aat taatga aga gta ctg ttc agt agg aga aca aga ctt ctt 719 Ile * Asn * * ArgVal Leu Phe Ser Arg Arg Thr Arg Leu Leu 225 230 235 gga ctt gat tag cttaac tct cag tga ttg gtg tta gag tac tgt tgt 767 Gly Leu Asp * Leu AsnSer Gln * Leu Val Leu Glu Tyr Cys Cys 240 245 250 tga gga tgg tta atttta taa tta agg gct ggg aat tgg gga gtt agt 815 * Gly Trp Leu Ile Leu *Leu Arg Ala Gly Asn Trp Gly Val Ser 255 260 ata tat tcc taa tcc taa ttatgt gca tct tta att tat gga ata act 863 Ile Tyr Ser * Ser * Leu Cys AlaSer Leu Ile Tyr Gly Ile Thr 265 270 275 ttg ttt ttt gtt tta act tct gataat ttg gat ttt ctg atg ttt aat 911 Leu Phe Phe Val Leu Thr Ser Asp AsnLeu Asp Phe Leu Met Phe Asn 280 285 290 gtg gtt ttg tct atc cct tat taacag tgc caa gct taa ggt ttt agc 959 Val Val Leu Ser Ile Pro Tyr * GlnCys Gln Ala * Gly Phe Ser 295 300 305 cat gct cca aaa tgg aat act tgtact gtt atg ttg ttc tgg tag tga 1007 His Ala Pro Lys Trp Asn Thr Cys ThrVal Met Leu Phe Trp * * 310 315 320 tgg tga tga aac ctg caa gtt atg tttatg tat aaa gcc act att gat 1055 Trp * * Asn Leu Gln Val Met Phe Met TyrLys Ala Thr Ile Asp 325 330 335 caa aat tag aga aat tat cat tta ata agtatc ctc cca tgt taa ttt 1103 Gln Asn * Arg Asn Tyr His Leu Ile Ser IleLeu Pro Cys * Phe 340 345 350 taa aaa aaa aaa aaa aaa 1121 * Lys Lys LysLys Lys 355 18 355 PRT Glycine max 18 Thr Arg Glu Thr Gly Gly Phe HisGly Tyr Arg Lys Leu Pro Asn Thr 1 5 10 15 Thr Ser Gly Leu Lys Leu SerVal Ser Asp Met Asn Met Asn Met Arg 20 25 30 Gln Gln Gln Val Ala Ser SerAsp Gln Asn Cys Ser Asn His Ser Ala 35 40 45 Ala Gly Glu Glu Asn Glu CysThr Val Arg Glu Gln Asp Arg Phe Met 50 55 60 Pro Ile Ala Asn Val Ile ArgIle Met Arg Lys Ile Leu Pro Pro His 65 70 75 80 Ala Lys Ile Ser Asp AspAla Lys Glu Thr Ile Gln Glu Cys Val Ser 85 90 95 Glu Tyr Ile Ser Phe IleThr Gly Glu Ala Asn Glu Arg Cys Gln Arg 100 105 110 Glu Gln Arg Lys ThrIle Thr Ala Glu Asp Val Leu Trp Ala Met Ser 115 120 125 Lys Leu Gly PheAsp Asp Tyr Ile Glu Pro Leu Thr Met Tyr Leu His 130 135 140 Arg Tyr ArgGlu Leu Glu Gly Asp Arg Thr Ser Met Arg Gly Glu Pro 145 150 155 160 LeuGly Lys Arg Thr Val Glu Tyr Ala Thr Leu Ala Thr Ala Phe Val 165 170 175Pro Pro Pro Phe His His His Asn Gly Tyr Phe Gly Ala Ala Met Pro 180 185190 Met Gly Thr Tyr Val Arg Glu Thr Pro Pro Asn Ala Ala Ser Ser His 195200 205 His His His Gly Ile Ser Asn Ala His Glu Pro Asn Ala Arg Ser Ile210 215 220 Asn Arg Val Leu Phe Ser Arg Arg Thr Arg Leu Leu Gly Leu AspLeu 225 230 235 240 Asn Ser Gln Leu Val Leu Glu Tyr Cys Cys Gly Trp LeuIle Leu Leu 245 250 255 Arg Ala Gly Asn Trp Gly Val Ser Ile Tyr Ser SerLeu Cys Ala Ser 260 265 270 Leu Ile Tyr Gly Ile Thr Leu Phe Phe Val LeuThr Ser Asp Asn Leu 275 280 285 Asp Phe Leu Met Phe Asn Val Val Leu SerIle Pro Tyr Gln Cys Gln 290 295 300 Ala Gly Phe Ser His Ala Pro Lys TrpAsn Thr Cys Thr Val Met Leu 305 310 315 320 Phe Trp Trp Asn Leu Gln ValMet Phe Met Tyr Lys Ala Thr Ile Asp 325 330 335 Gln Asn Arg Asn Tyr HisLeu Ile Ser Ile Leu Pro Cys Phe Lys Lys 340 345 350 Lys Lys Lys 355 19796 DNA Glycine max CDS (1)...(513) 19 gca cga gca atg gcg gga gtg agggaa cag gac cag tac atg ccg ata 48 Ala Arg Ala Met Ala Gly Val Arg GluGln Asp Gln Tyr Met Pro Ile 1 5 10 15 gcg aac gtg ata agg atc atg cgtcgg att ctg cca gcg cac gcg aag 96 Ala Asn Val Ile Arg Ile Met Arg ArgIle Leu Pro Ala His Ala Lys 20 25 30 atc tca gac gac gcg aag gag acg atccag gag tgc gtg tct gag tac 144 Ile Ser Asp Asp Ala Lys Glu Thr Ile GlnGlu Cys Val Ser Glu Tyr 35 40 45 atc agt ttc atc acg gcg gag gcg aac gagcgg tgc cag cgg gag cag 192 Ile Ser Phe Ile Thr Ala Glu Ala Asn Glu ArgCys Gln Arg Glu Gln 50 55 60 cgg aag acg gtg acc gca gag gat gtg ttg tgggcg atg gag aag ctt 240 Arg Lys Thr Val Thr Ala Glu Asp Val Leu Trp AlaMet Glu Lys Leu 65 70 75 80 ggc ttt gac aac tac gct cac cct ctc tct ctttac ctt cac cgc tac 288 Gly Phe Asp Asn Tyr Ala His Pro Leu Ser Leu TyrLeu His Arg Tyr 85 90 95 cgc gag agt gaa gga gaa cct gct tct gtc aga cgcgct tct tct gca 336 Arg Glu Ser Glu Gly Glu Pro Ala Ser Val Arg Arg AlaSer Ser Ala 100 105 110 atg ggg atc aat aat aat atg gtg cac cca cct tatatt aat tct cat 384 Met Gly Ile Asn Asn Asn Met Val His Pro Pro Tyr IleAsn Ser His 115 120 125 ggc ttt gga atg ttt gat ttt gac cca tca tcg caaggg ttt tac agg 432 Gly Phe Gly Met Phe Asp Phe Asp Pro Ser Ser Gln GlyPhe Tyr Arg 130 135 140 gac gat cat aac gct gct tct gga tct ggt ggt tttgtt gcg cct ttt 480 Asp Asp His Asn Ala Ala Ser Gly Ser Gly Gly Phe ValAla Pro Phe 145 150 155 160 gat cct tat gct aac atc aaa cgt gat gcc ctgtgatcatgta agaacaacaa 533 Asp Pro Tyr Ala Asn Ile Lys Arg Asp Ala Leu165 170 ctagtgcatg ctgctttttc acttggttag ttatattcaa gcacaagcacatgcaggtgc 593 agctgcaact atttagcttc atctacaaat cttttttcct ctcttcttctcatgctttaa 653 ttatttagag acaatacttg ttattcattg ttatgctcaa ttgctagcttctattcatcg 713 tcgactgtct gtattgttga tgttcattac agtaacagat aagatggtaactgctttact 773 acttcaaaaa aaaaaaaaaa aaa 796 20 171 PRT Glycine max 20Ala Arg Ala Met Ala Gly Val Arg Glu Gln Asp Gln Tyr Met Pro Ile 1 5 1015 Ala Asn Val Ile Arg Ile Met Arg Arg Ile Leu Pro Ala His Ala Lys 20 2530 Ile Ser Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys Val Ser Glu Tyr 35 4045 Ile Ser Phe Ile Thr Ala Glu Ala Asn Glu Arg Cys Gln Arg Glu Gln 50 5560 Arg Lys Thr Val Thr Ala Glu Asp Val Leu Trp Ala Met Glu Lys Leu 65 7075 80 Gly Phe Asp Asn Tyr Ala His Pro Leu Ser Leu Tyr Leu His Arg Tyr 8590 95 Arg Glu Ser Glu Gly Glu Pro Ala Ser Val Arg Arg Ala Ser Ser Ala100 105 110 Met Gly Ile Asn Asn Asn Met Val His Pro Pro Tyr Ile Asn SerHis 115 120 125 Gly Phe Gly Met Phe Asp Phe Asp Pro Ser Ser Gln Gly PheTyr Arg 130 135 140 Asp Asp His Asn Ala Ala Ser Gly Ser Gly Gly Phe ValAla Pro Phe 145 150 155 160 Asp Pro Tyr Ala Asn Ile Lys Arg Asp Ala Leu165 170 21 1098 DNA Triticum aestivum CDS (55)...(894) 21 gcacgagcaagtgcgagtgc gactacctgc attgcacctt ggctagccct agac atg 57 Met 1 gag aacgac ggc gtc ccc aac gga cca gcg gcg ccg gca cct acc cag 105 Glu Asn AspGly Val Pro Asn Gly Pro Ala Ala Pro Ala Pro Thr Gln 5 10 15 ggg acg ccggtg gtg cgg gag cag gac cgg ctg atg ccg atc gcg aac 153 Gly Thr Pro ValVal Arg Glu Gln Asp Arg Leu Met Pro Ile Ala Asn 20 25 30 gtg atc cgc atcatg cgc cgt gcg ctc cct gcc cac gcc aag atc tcc 201 Val Ile Arg Ile MetArg Arg Ala Leu Pro Ala His Ala Lys Ile Ser 35 40 45 gac gac gcc aag gaggcg att cag gaa tgc gtg tcc gag ttc atc agc 249 Asp Asp Ala Lys Glu AlaIle Gln Glu Cys Val Ser Glu Phe Ile Ser 50 55 60 65 ttc gtc acc ggc gaggcc aac gaa cgg tgc cgc atg cag cac cgc aag 297 Phe Val Thr Gly Glu AlaAsn Glu Arg Cys Arg Met Gln His Arg Lys 70 75 80 acc gtc aac gcc gaa gacatc gtg tgg gcc cta aac cgc ctc ggc ttc 345 Thr Val Asn Ala Glu Asp IleVal Trp Ala Leu Asn Arg Leu Gly Phe 85 90 95 gac gac tac gtc gtg ccc ctcagc gtc ttc ctg cac cgc atg cgc gac 393 Asp Asp Tyr Val Val Pro Leu SerVal Phe Leu His Arg Met Arg Asp 100 105 110 ccc gag gcg ggg aca ggt ggtgcc gct gca ggc gac agc cgc gcc gtg 441 Pro Glu Ala Gly Thr Gly Gly AlaAla Ala Gly Asp Ser Arg Ala Val 115 120 125 acg agt gcg cct ccc cgc gcggcc ccg ccc gtg atc cac gcc gtg ccg 489 Thr Ser Ala Pro Pro Arg Ala AlaPro Pro Val Ile His Ala Val Pro 130 135 140 145 ctg cag gct cag cgc ccgatg tac gcg ccc ccg gct ccg ttg cag gtt 537 Leu Gln Ala Gln Arg Pro MetTyr Ala Pro Pro Ala Pro Leu Gln Val 150 155 160 gag aat cag atg cag cggcct gtg tac gct ccc ccg gct ccg gtg cag 585 Glu Asn Gln Met Gln Arg ProVal Tyr Ala Pro Pro Ala Pro Val Gln 165 170 175 gtt cag atg cag cgg ggcatc tat ggg ccc cgg gct cca gtg cac ggg 633 Val Gln Met Gln Arg Gly IleTyr Gly Pro Arg Ala Pro Val His Gly 180 185 190 tac gcc gtc gga atg gcgccc gtg cgg gcc aac gtc ggc ggg cag tac 681 Tyr Ala Val Gly Met Ala ProVal Arg Ala Asn Val Gly Gly Gln Tyr 195 200 205 cag gtg ttc ggc gga gagggt gtc atg gcc cag caa tac tac ggg tac 729 Gln Val Phe Gly Gly Glu GlyVal Met Ala Gln Gln Tyr Tyr Gly Tyr 210 215 220 225 ggg tac gag gaa ggagcg tac ggc gca ggt agc agc aac gga gga gcc 777 Gly Tyr Glu Glu Gly AlaTyr Gly Ala Gly Ser Ser Asn Gly Gly Ala 230 235 240 gcc att ggc gac gaggag agc tcg tcc aac ggc gtg ccg gca ccg ggg 825 Ala Ile Gly Asp Glu GluSer Ser Ser Asn Gly Val Pro Ala Pro Gly 245 250 255 gag ggc atg ggg gagcca gag cca gag cca gca gca gaa gaa tcg cat 873 Glu Gly Met Gly Glu ProGlu Pro Glu Pro Ala Ala Glu Glu Ser His 260 265 270 gac aag ccc gtc caatct ggc tagtcgcgtg cgcggcgcgc gttagcttct 924 Asp Lys Pro Val Gln Ser Gly275 280 gcgtcctgtg tactgtaata atttgccgtg tcgatccggc catggtttgtgtgtgcgtag 984 tgcttatcta atgtgggctt gtcctctagt aattcatgta ttgcttatctaatgtggact 1044 tgtcctctag taattcatgt actctttgct gttgaaaaaa aaaaaaaaaaaaaa 1098 22 280 PRT Triticum aestivum 22 Met Glu Asn Asp Gly Val ProAsn Gly Pro Ala Ala Pro Ala Pro Thr 1 5 10 15 Gln Gly Thr Pro Val ValArg Glu Gln Asp Arg Leu Met Pro Ile Ala 20 25 30 Asn Val Ile Arg Ile MetArg Arg Ala Leu Pro Ala His Ala Lys Ile 35 40 45 Ser Asp Asp Ala Lys GluAla Ile Gln Glu Cys Val Ser Glu Phe Ile 50 55 60 Ser Phe Val Thr Gly GluAla Asn Glu Arg Cys Arg Met Gln His Arg 65 70 75 80 Lys Thr Val Asn AlaGlu Asp Ile Val Trp Ala Leu Asn Arg Leu Gly 85 90 95 Phe Asp Asp Tyr ValVal Pro Leu Ser Val Phe Leu His Arg Met Arg 100 105 110 Asp Pro Glu AlaGly Thr Gly Gly Ala Ala Ala Gly Asp Ser Arg Ala 115 120 125 Val Thr SerAla Pro Pro Arg Ala Ala Pro Pro Val Ile His Ala Val 130 135 140 Pro LeuGln Ala Gln Arg Pro Met Tyr Ala Pro Pro Ala Pro Leu Gln 145 150 155 160Val Glu Asn Gln Met Gln Arg Pro Val Tyr Ala Pro Pro Ala Pro Val 165 170175 Gln Val Gln Met Gln Arg Gly Ile Tyr Gly Pro Arg Ala Pro Val His 180185 190 Gly Tyr Ala Val Gly Met Ala Pro Val Arg Ala Asn Val Gly Gly Gln195 200 205 Tyr Gln Val Phe Gly Gly Glu Gly Val Met Ala Gln Gln Tyr TyrGly 210 215 220 Tyr Gly Tyr Glu Glu Gly Ala Tyr Gly Ala Gly Ser Ser AsnGly Gly 225 230 235 240 Ala Ala Ile Gly Asp Glu Glu Ser Ser Ser Asn GlyVal Pro Ala Pro 245 250 255 Gly Glu Gly Met Gly Glu Pro Glu Pro Glu ProAla Ala Glu Glu Ser 260 265 270 His Asp Lys Pro Val Gln Ser Gly 275 28023 65 PRT Artificial Sequence LEC1 consensus protein sequence 23 Arg GluGln Asp Xaa Xaa Met Pro Ile Ala Asn Val Ile Arg Ile Met 1 5 10 15 ArgXaa Xaa Leu Pro Xaa His Ala Lys Ile Ser Asp Asp Ala Lys Glu 20 25 30 XaaIle Gln Glu Cys Val Ser Glu Tyr Ile Ser Phe Xaa Thr Xaa Glu 35 40 45 AlaAsn Xaa Arg Cys Xaa Xaa Xaa Xaa Arg Lys Thr Xaa Xaa Xaa Glu 50 55 60 Xaa65 24 36 DNA Artificial Sequence Sal-A20 oligo 24 tcgacccacg cgtccgaaaaaaaaaaaaaa aaaaaa 36 25 22 DNA Artificial Sequence primer-forward 25cgctctgtca cctgttgtac tc 22 26 22 DNA Artificial Sequence primer-reverse26 cgtgatgaag ctgatgtact cc 22

What is claimed is:
 1. A method for enhancing tissue culture response ina plant cell comprising introducing into the plant cell at least oneLEC1 polypeptide or at least one LEC1 polynucleotide.
 2. The method ofclaim 1 wherein the plant cell is transformed with at least one LEC1polynucleotide.
 3. The method of claim 1 wherein at least onepolynucleotide is operably linked to a promoter driving expression inthe plant cell and growing the plant cell.
 4. The method of claim 1wherein the plant cell is a recalcitrant cell.
 5. The method of claim 1wherein the plant cell is an inbred plant cell.
 6. A method for inducingsomatic embryogenesis in a plant cell comprising introducing into aplant cell at least one LEC1 polypeptide or at least one LEC1polynucleotide, wherein the plant cell is transformed and grown toproduce a transformed embryo and wherein the plant cell is not anArabidopsis cell.
 7. The method of claim 6 wherein the plant cell istransformed with at least one LEC1 polynucleotide.
 8. The method ofclaim 6 wherein at least one polynucleotide is operably linked to apromoter driving expression in the plant cell.
 9. The method of claim 6further comprising growing the transformed embryo under plant growingconditions to produce a regenerated plant.
 10. A transgenic plantproduced by the method of claim
 9. 11. The method of claim 6 wherein theplant cell is from corn, soybean, sorghum, wheat, rice, alfalfa,sunflower, canola or cotton.
 12. A method for positive selection of atransformed cell comprising introducing into a plant cell at least oneLEC1 polynucleotide or at least one LEC1 polypeptide and growing thetransformed plant cell, wherein the plant cell is transformed and grownunder conditions sufficient to induce embryogenesis to provide apositive selection means.
 13. The method of claim 12 further comprisingaltering media components to favor the growth of transformed plantcells.
 14. The method of claim 13 wherein the media components arealtered to reduce somatic embryogenesis in non-transformed cells. 15.The method of claim 12 wherein the plant cell is transformed with atleast one LEC1 polynucleotide.
 16. The method of claim 15 wherein atleast one LEC1 polynucleotide is operably linked to a promoter drivingexpression in a plant.
 17. The method of claim 16 wherein thepolynucleotide is excised.
 18. The method of claim 15 wherein thepolynucleotide is flanked by FRT sequences to allow FLP mediatedexcision of the polynucleotide.
 19. A method for inducing apomixis in aplant cell comprising introducing into a responsive plant cell at leastone LEC1 polypeptide or at least one LEC1 polynucleotide and growing theplant cell, wherein the introducing and growing is done under conditionssufficient to produce a transformed somatic embryo.
 20. The method ofclaim 19 wherein the plant cell is transformed with at least one LEC1polynucleotide.
 21. The method of claim 19 wherein the at least onepolynucleotide is expressed in integument or nucellus tissue.
 22. Themethod of claim 19 wherein the at least one polynucleotide is operablylinked to a promoter driving expression in a plant cell
 23. The methodof claim 22 further comprising suppressing in the plant cell expressionof an FIE polycomb polynucleotide.
 24. The method of claim 22 whereinthe promoter is an inducible promoter.
 25. The method of claim 19further comprising growing the transformed somatic embryo under plantgrowing conditions to produce a regenerated plant.
 26. A plant producedby the method of claim
 25. 27. The plant of claim 26, wherein the plantis male sterile.
 28. A method for increasing transformation efficiencycomprising introducing at least one LEC1 polypeptide or at least oneLEC1 polynucleotide into a plant cell.
 29. The method of claim 28wherein the plant cell is transformed with at least one LEC1polynucleotide.
 30. The method of claim 29 wherein plant cell is incontact with medium that retards growth of somatic embryo growth innon-transformed plants.
 31. The method of claim 29 whereintransformation is conducted with reduced levels of auxin or no auxin.32. The method of claim 29 wherein the at least one polynucleotide isoperably linked to a promoter driving expression in a plant cell
 33. Themethod of claim 28 wherein the plant cell is a recalcitrant cell. 34.The method of claim 28 wherein the plant cell is an inbred cell.
 35. Amethod for increasing recovery of regenerated plants comprisingintroducing into a plant cell at least one LEC1 polypeptide or at leastone LEC1 polynucleotide and growing the plant cell.
 36. The method ofclaim 35 wherein the plant cell is transformed with at least one LEC1polynucleotide.
 37. The method of claim 35 wherein the at least onepolynucleotide is operably linked to a promoter driving expression in aplant cell
 38. The method of claim 35 wherein the plant cell is arecalcitrant cell.
 39. The method of claim 35 wherein the plant cell isan inbred plant cell.